Stable immunogenic product comprising antigenic heterocomplexes

ABSTRACT

The invention relates to a stable immunogenic product for the induction of antibodies against one or more antigenic proteins in a subject. The invention is characterised in that it comprises proteinaceous immunogenic heterocomplexes which are formed by associations between (i) antigenic protein molecules and (ii) proteinaceous carrier molecules and in that less than 40% of the antigenic proteins (i) are linked to the proteinaceous carrier molecules (ii) by a covalent bond.

FIELD OF THE INVENTION

The present invention relates to stable immunogenic products comprisingimmunogenic protein heterocomplexes for obtaining a humoral immuneresponse with production of specific antibodies raised against one oremore antigens, in particular against a <<self>> antigen, as well astheir use in the field of vaccines.

PRIOR ART

Obtaining a high level antibody response from a given antibody, in anindividual, is an object commonly sought, whether the antigen is a<<foreign>> antigen or a <<self>> antigen.

However, the problem of a good recognition of the antigen against whichan antibody response is being sought, in an individual, should be solvedin a number of cases, more particularly (a) when the antigen of interestbehaves like a <<hapten>>, i.e. a low molecular mass chemical structurewhich is little or not immunogenic under a free form, but that, oncefixed on a high molecular mass molecule, is able to induce theproduction of specific antibodies of such a hapten, and (b) when theantigen of interest is a self protein, i.e. a protein being naturallyproduced in the individual, for which there exists an immune tolerancedue to the deletion of corresponding lymphocyte T clones, during thedevelopment of the immune system.

In order to cause, or increase, the recognition of an antigen ofinterest by B cells, various immunogenic constructions were developed inthe state of the art.

A first immunogenic construction form comprises a covalent coupling ofthe antigen of interest on a carrier molecule, the carrier moleculebringing structures recognized by the auxiliary T lymphocytes (<<Thelper>> cells), in association with class II molecules of theHistocompatibility Major Complex (HMC), and activating the auxiliary Tlymphocytes then producing various cytokins, amongst which IL-2, saidcytokins activating in turn the specific B cell clones of the antigen ofinterest. The specific B cells of the antigen of interest, onceactivated, multiply and produce antibodies specific to the antigen ofinterest, which is the objective being sought. Generally, such a type ofimmunogenic constructions comprises products of the covalent chemicalcoupling between the antigen of interest and the carrier molecule,which, after purification and removal steps of the non coupled products,are final products with a well defined chemical structure.

The first form of an immunogenic construction is for example illustratedby the article by Richard and al. describing the preparation of productsof the covalent coupling between IL-9 and ovalbumin (Proc. Natl. Acad.Sci. USA, Vol. 97(2): 767-772). It is also illustrated in such U.S. Pat.No. 6,340,461 (Terman) which discloses coupling products between one ormore copies of an antigen of interest, against which a specific antibodyresponse is being sought in an individual, and a carrier moleculeconsisting in a <<Superantigen>>. The antigen of interest is coupledexclusively covalently to the carrier molecule, for example, by means ofglutaraldehyde (also called <<pentanedial>>), the non covalently coupledproducts being removed in order to obtain a chemically well definedfinal product.

Optionally, the product from the covalent coupling between the antigenof interest and the superantigen could be prepared in the form of apolymer of said coupling product, for example, through a non covalentbond of the monomeric coupling products between one another, throughionic interactions, adsorption interactions as well as biospecificinteractions. For example, the monomeric coupling products could formcomplexes with highly positively or negatively charged molecules,through salt bridges produced in low ionic strength conditions. Largescomplexes of monomeric coupling products are prepared using chargedpolymers such as poly(L-glutamic acid) or poly(L-lysine) polymers.According to another embodiment of a monomeric coupling product polymer,the exclusively covalent coupling products between the antigen ofinterest and the superantigen could be adsorbed or coupled noncovalently at the surface of microparticles, such as latex beads orother hydrophobic polymers.

A second embodiment of such immunogenic constructions commonly called<<MAP>> structure (for <<Multi-Antigenic Protein>>) generally have theform of a protein backbone comprised of a linear or branched,poly(lysine) polymer, onto which one or more antigens of interest arecovalently bound.

A third embodiment of such immunogenic constructions consists inmicroparticles onto which fixed the antigen(s) of interest is/are bound.Various forms of antigen carrier microparticles are known.

Are known, for example, iscomes (for <<immunostimulating complexes>>)comprised of an antigenic complex and an adjuvant, the QuiIA compound.

Liposomes are also known having the same inconvenients as the iscomes,i.e. more particularly some toxicity and immunological side effects, dueto their lack of purity.

Biodegradable microparticles are also known such as lactic acid andglutamic acid polymers (Aguado and Lambert, 1992, Immuno. Biol., Vol.184: 113-125) as well as starch particles (US Patent Application2002/0098203—Gutavsson et al.), in the polymeric matrix of whichantigens of interest are trapped. Such particles release the antigenunder their soluble form during the degradation of the polymeric matrix.

Particles have also been disclosed exclusively comprised of hybridrecombinant proteins, as disclosed in French Patent Application FR2,635,532 (Thiollais et al.).

Porous microspheres are also known wherein the antigens are immobilizedwithin micropores through captation or physical coupling, as disclosedin the U.S. Pat. No. 5,008,116 (Cahn).

However, the various solutions suggested in the state of the art sharein common at least one technical inconvenient related to theirpreparation method, i.e. the loss of a high proportion of the antigenicmaterial of interest, due to a necessary step for removing the noncoupled or non adsorbed antigens.

Moreover, while the prior art techniques allow to provide an associationbetween the low molecular mass antigen of interest with a carriermolecule, they are generally not adapted to coupling a high molecularmass antigen of interest, for example, of more than 10 kDa, with thecarrier molecule, because, in particular, of steric hindrancespreventing coupling a high number of molecules of antigens of interesthaving a high molecular mass with an identical carrier molecule.

Finally, most if not all the known peptide antigenic constructionsencompass in their structure a single carrier molecule, which is atechnical inconvenient when the objective is to induce a preventive ortherapeutic immune response both against the antigen of interest and thecarrier molecule itself.

There is therefore a need in the state of the art for improvedimmunogenic constructions allowing for the production of a high level ofantibodies specific to an antigen of interest in an individual wheresuch a humoral immune response is sought, being less expensive, simpleto prepare and able to be synthetized reproducibly.

SUMMARY OF THE INVENTION

The present invention provides new immunogenic constructions allowing tosolve the various technical problems encountered with the immunogenicconstructions as known in the prior art and allowing to meet theabove-described various technical needs.

The object of the invention is to provide a stable immunogenic productfor inducing antibodies raised against one or more antigenic proteins ina subject, characterized in that it comprises protein immunogenicheterocomplexes consisting of associations between (i) antigenic proteinmolecules and (ii) carrier protein molecules and in that less than 40%of the antigenic proteins (i) are covalently linked to carrier proteinmolecules (ii).

Another object of the invention is also an immunogenic productcomprising stable protein immunogenic heterocomplexes for inducingantibodies raised against one or more antigenic proteins in a subject,each heterocomplex comprising (i) a plurality of antigenic proteins,linked to a (ii) carrier protein molecule, characterized in that lessthan 40% of the antigenic proteins (i) are covalently linked to carrierprotein molecules (ii).

Preferably, the immunogenic heterocomplex making up the immunogenicproduct according to the invention comprises 5 to 50 antigenic proteins(i) for one carrier protein molecule (ii), preferably 20 to 40 antigenicproteins (i) for one carrier protein molecule (ii).

Preferably, the covalent bonds between one or more antigenic proteins(i) and the carrier protein molecules (ii) occur by means of afunctional binding chemical agent.

It is meant under antigenic molecule of interest, any protein comprisingone or more B epitopes of a native antigenic protein against which theproduction of antibodies is being sought. Said antigenic molecule ofinterest can consist in the native protein itself or a protein derivateof the native protein, such as a peptide fragment of the native protein,as well as any biologically inactivated form of the native proteinobtained through chemical, physical treatment or genetic mutation. Theantigenic molecule of interest could also consist in a homo-oligomer ora homo-polymer of the native protein as well as a homo-oligomer or ahomo-polymer of a peptide fragment of the native protein. The antigenicprotein of interest could also consist in a hetero-oligomer or ahetero-polymer comprising a combination of several distinct peptidefragments initially included in the native protein.

According to the general embodiment of an immunogenic product accordingto the invention, the carrier protein molecule (ii) is an immunogenicprotein inducing the production of T helper lymphocytes and/or ofcytotoxic T lymphocytes raised against cells having at their surfacesaid carrier protein molecule or any peptide being derived therefrom, inassociation with presenting molecules of the Major HistocompatibilityComplex (MHC), respectively of class I and/or class II. The carrierprotein molecule (ii) could also be an immunogenic protein inducing boththe production of T helper lymphocytes and the production of antibodiesby B lymphocytes raised against the carrier protein.

According to an embodiment of a particular interest, the immunogenicproduct is characterized in that the carrier protein molecule (ii) is animmunogenic protein inducing the production of T cytotoxic lymphocytesraised against cells having at their surface said carrier proteinmolecule or any peptide being derived therefrom, in association withmolecules of the Major Histocompatibility Complex (MHC) class I.

The preferred immunogenic products according to the invention areselected amongst immunogenic products comprising the followingheterocomplexes, wherein the antigenic proteins (i), on the one hand,and the protein carrier molecule (ii), on the other hand, arerespectively:

a) (i) IL-4 and (ii) KLH;

b) (i) alpha interferon and (ii) KLH;

c) (i) VEGF and (ii) KLH;

d) (i) IL-10 and (ii) KLH;

e) (i) alpha interferon and (ii) gp 160 of VIH1

f) (i) IL-4 and (ii) the Bet v 1 allergenic antigen; and

g) (i) VEGF and (ii) the papillomavirus E7 protein;

h) (i) ) the inactivated VIH1 Tat protein and (ii) the VIH1 gp 120protein.

i) (i) an IgE isotype human antibody and (ii) the inactivated VIH1 Tatprotein;

j) (i) the ricin β fragment and (ii) KLH.

The invention also relates to a composition, more particularly, apharmaceutical composition, an immunogenic composition or a vaccinecomposition, characterized in that it comprises an immunogenic productsuch as hereinabove described.

It also relates to a method for preparing an immunogenic productaccording to any one of claims 1 to 16, characterized in that itcomprises the following steps of:

a) incubating the antigenic proteins (i) and the carrier molecule (ii)in a molar ratio (i):(ii) ranging from 10:1 to 50:1 in the presence of abinding chemical; and

b) collecting the immunogenic product comprising immunogenicheterocomplexes being prepared in step a).

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the characterization of the immunogenic productcomprising murine KLH-VEGF heterocomplexes through isoelectrofocusing inan agarose gel followed by the emergence of proteins throughimmuno-blotting (<<Western Blot>>).

FIG. 2 illustrates the characterization of the immunogenic productcomprising human KLH-VEGF heterocomplexes through isoelectrofocusingthrough a colouration with Coomassie blue, followed by an immunoblotting(<<Western Blot>>). The isoelectrofocusing gel is represented at theleft of the figure. The immunoblotting gels using anti-KLH (left) orhuman anti-VEGF (right) antibodies are illustrated on the right of thefigure.

FIG. 3 illustrates the characterization of the immunogenic productcomprising human KLH-IL4 heterocomplexes through isoelectrofocusing inan agarose gel followed by the emergence of proteins throughimmunoblotting (<<Western Blot>>).

FIG. 4 illustrates the characterization of the immunogenic productcomprising gp 160-IFNα complexes through isoelectrofocusing in anagarose gel followed by the mergence of proteins through immunoblotting(<<Western Blot>>).

FIG. 5 illustrates the immunogenic (humoral) activity of the murineKLH-VEGF immunogenic product through determination of the title antibodyobtained after an immunization of mice. FIG. 5A relates to miceimmunized with murine VEGF. FIG. 5B relates to mice immunized with theimmunogenic product comprising KLK-VEGF heterocomplexes. FIG. 5Cillustrates control mice injected with Freund's Incomplete Adjuvant(FIA).

FIG. 6 shows the immunogenic (humoral) activity of the murin KLH-VEGFimmunogenic product, through determination of the neutralizing power ofantibodies obtained after immunization, towards the angiogenic activityof the VEGF protein.

FIG. 7 illustrates the immunogenic (humoral) activity of the humanKLH-VEGF immunogenic product, through determination of the antibodytitle obtained after immunization of mice.

FIG. 8 illustrates the immunogenic (humoral) activity of the humanKLH-VEGF immunogenic product, through determination of the neutralizingpower of antibodies obtained after immunization, towards the angiogenicactivity of the VEGF protein, measured through the proliferation ofendothelial cells.

FIG. 9 illustrates the immunogenic (humoral) activity of the murineKLH-IL4 immunogenic product, through determination of the title antibodyobtained after immunization.

FIG. 10 illustrates the immunogenic (humoral) activity of the murineKLH-IL4 immunogenic product, through determination of the neutralizingpower of antibodies obtained after immunization, towards the inducingactivity of the proliferation of HT-2 cells by the IL4.

FIG. 11 illustrates the results of the production of the IgG and IgEclass antibodies raised against Bet v 1, after the injection ofbirch-tree pollen, to mice preliminarily immunized with an immunogenicproduct according to the invention comprising KLH-IL4 complexes.

FIG. 12 illustrates the immunogenic (humoral) activity of the humanKLH-IL4 immunogenic product through determination of the neutralizingpower of antibodies obtained after immunization, towards the inducingactivity of the proliferation of HT-2 cells by the ILA.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides new immunogenic constructions inducing a highlevel of production of antibodies specific to an antigen of interest, inan individual.

The Immunogenic Protein Heterocomplexes According to the Invention

It has been shown according to the invention that the production of ahigh level of antibodies specific to an antigen of interest could beobtained, in an individual, through the immunization of such anindividual with an immunogenic product where said antigen of interest isassociated with a carrier protein molecule, the association between saidantigen of interest and said carrier protein being partially covalentand partially non covalent.

More specifically, it has been shown according to the invention that anexcellent antibody response raised against an antigen of interest isobtained when an individual is being immunized with a stable immunogenicproduct comprising protein heterocomplexes, wherein the heterocomplexescomprise stable associations between antigen of interest and saidcarrier protein molecule and wherein only a low proportion of suchassociations is due to a covalent bond between the antigen of interestand the carrier protein molecule, the other associations between theantigen of interest and the carrier protein molecule being produced byweak bonds, ionic interactions, hydrogen bonds, Van der Waals forces,etc.

In particular, it has been shown according to the invention that anoptimum antibody response is reached when, in a stable immunogenicproduct such as described hereinabove, less than 40% of the molecules ofthe antigen of interest are covalently linked to the carrier proteinmolecules. According to the invention, an antigenic molecule of interestis covalently linked to a carrier protein molecule by <<one>> covalentbond means that said molecule of antigen of interest is covalentlylinked, chemically, to said carrier protein molecule, by at least onecovalent bond, i.e. optionally by two covalent bonds or more.

The percentage of carrier protein molecules and of antigenic proteinproteins of interest linked between one another through covalent bondsin an immunogenic product of the invention can be easily checked by theman of the art.

For example, determining the percentage of antigenic molecules ofinterest linked to the carrier protein molecules through a covalent linkin an immunogenic product of the invention could be made using thefollowing steps of:

(i) submitting said immunogenic product in solution to denaturing andreducing conditions;

(ii) performing a size exclusion chromatography step with the product asobtained at the end of step (ii) during which the various proteincomponents with decreasing molecular mass are successively eluted fromthe size exclusion chromatography support;

(iii) measuring the amount of antigen of interest linked through acovalent bond to the carrier molecule in the eluate fraction containingthe protein components with the highest molecular mass;

(iv) comparing the amount of antigen of interest measured in step (iii)with the total amount of antigen of interest initially included in thestarting immunogenic product.

In step (i) of the method for determining the above-described covalentlink percentage, incubating a given amount (in number of moles or inweight) of the immunogenic product of the invention under denaturing andreducing conditions leads to a disassociation of the weak bonds betweenthe various protein components not linked between one another through acovalent bond.

Amongst preferred denaturing conditions there is the presence of urea,for example, in the final 8M concentration, or the presence of SDS, forexample, in the 1% final concentration in total weight of the solutioncontaining the immunogenic product. Amongst preferred reducingconditions there is the presence of β-mercaptoethanol, for example inthe 5% final concentration of the total volume of the solutioncontaining the immunogenic product.

In step (ii) of the method for determining the percentage of moleculesof antigen of interest and molecule of carrier protein linked betweenone another through covalent bonds, the size exclusion chromatographysupport is selected by the man of the art according to his technicalgeneral knowledge. For example, the man of the art could make use ofchromatographic supports as marketed by the Pharmacia Corporation underthe <<Superdex 75™>> and <<Superdex 200™>> trade marks.

In step (ii), the molecular fraction corresponding to the carriermolecule covalently linked to the molecules of antigen of interest iseluted first, before the eluate fraction(s) containing the antigen ofinterest under a free form. The antigen of interest being eluted under afree form corresponds to the fraction of the antigen of interest, whichwas not covalently linked to the carrier molecule, within the startingimmunogenic product. It is on the high molecular mass protein fractionthat occurs the measurement of the amount of the antigen of interestcovalently linked to the carrier protein molecule, for example, in animmuno-enzyme test, in a radioimmunologic test or in animmunofluorescence test, either direct or indirect (<<sandwich>>), usingantibodies specific to the antigen of interest and which do not have anyimmunologic reaction crossed with the carrier protein molecule.

In step (iii), the amount of the antigen of interest covalently linkedwith the carrier protein molecule, being measured as describedhereinabove, is compared with the initial amount of the antigen ofinterest being included in the given amount (in number of moles or inweight) of the starting immunogenic product and the percentage of theantigen of interest is thereby calculated, which is covalently linked tothe carrier protein molecule, in the immunogenic product of theinvention.

The percentage of carrier protein molecules and of antigenic proteinproteins of interest linked between one another through covalent bonds,in an immunogenic product of the invention, can be easily checked by theman of the art, making use of a second method comprising the followingsteps of:

a) immobilizing on a support of specifically antibodies raised againstthe carrier protein;

b) brinding into contact the antibodies raised against the carrierprotein, which were immobilized on the support in step a), with a knownamount of molecules of the immunogenic product to be tested comprisingsaid carrier protein and an antigenic protein of interest;

c) removing the molecules of the immunogenic product which are notlinked to the anti-carrier protein antibodies immobilized in step a), bymeans of a buffering aqueous solution comprising one ore more proteindenaturing agents;

d) d1) bringing into contact (i) immunogenic complexes formed in step c)between the immobilized anti-carrier protein antibodies and themolecules of the immunogenic product with (ii) antibodies specificallyraised against the carrier protein;

d2) separately from step d1), briging into contact the immunogeniccomplexes formed in step c) between the immobilized anti-carryingprotein antibodies and the molecules of the immunogenic product with(ii) antibodies specifically raised against the antigenic protein ofinterest;

e) e1) quantifying the antibodies added in step d1) having been linkedto the carrier protein;

e2) quantifying the antibodies added in step d2) having been linked tothe antigenic protein;

f) calculating the ratio between:

(i) the amount of anti-carrier protein bound antibodies measured in stepe1); and

(ii) the amount of anti-carrier protein bound antibodies measured instep e2),

said ratio consisting in the proportion of carrier protein molecules andantigenic protein molecules of interest being linked between one anotherthrough covalent bonds, within the starting immunogenic product.

In step c) of the above described method, the use of an aqueous washingsolution containing one or more protein denaturing agents leads to adenaturation of the immunogenic product linked to the anti-carrierprotein antibodies, resulting in the release, in the washing solution,of antigenic protein molecules of interest which are not covalentlylinked to the carrier protein molecules. Therefore, in step d2) of themethod, only the antigenic protein molecules of interest beingcovalently linked to the carrier protein molecules are quantified.

Preferably, the denaturing buffering solution used in step c) contains asurfactant such as Tween®20, in a final concentration of 0.1% v/v.

in steps d1) and d2), the amounts of bound antibodies are preferablymeasured through incubating antigen-antibodies complexes formed at theend of each of said steps with a new antibody being labelled through adetectable molecule, respectively:

(i) in step d1), a new antibody directed against an the anti-carrierprotein antibody and labelled with a detectable molecule;

(ii) in step d2), a new antibody directed against an antibodyanti-antigeni protein of interest and labelled with a detectablemolecule.

The detectable molecule is indiscriminately either a radioactivemolecule, a fluorescent molecule or an enzyme. As an enzyme, peroxydasecould more particularly be used, its presence being revealed throughcolorimetry, after incubation with the ortho-phenylenediamine (OPD)substrate.

A detailed protocol of the above-mentioned method is described in theexamples.

By way of illustration, it has been shown according to the invention,using the first or the second above described quantification methodsthat:

in the immunogenic product comprising heterocomplexes between the KLHcarrier molecule and human alpha interferon molecules, from 3 to 8% ofthe alpha interferon molecules are covalently linked to the KLH carrierprotein molecule;

in the immunogenic product comprising the heterocomplexes between theKLH carrier protein molecule and murine IL-4 molecules, about 11% of theIL-4 molecules are covalently linked to the KLH carrier proteinmolecule.

Obviously, depending on the preparations, the percentage of molecules ofantigenic protein of interest covalently linked to the carrier proteinmolecules could significantly vary. However, in all cases, such apercentage is always lower than 40%.

The object of the invention is to provide a stable immunogenic productfor inducing antibodies raised against one or more antigenic proteins ina subject, characterized in that it comprises protein immunogenicheterocomplexes comprising associations between (i) antigenic proteinmolecules and (ii) carrier protein molecules and in that less than 40%of the antigenic proteins (i) are covalently linked to carrier proteinmolecules (ii).

Another object of the invention is also to provide an immunogenicproduct comprising stable protein immunogenic heterocomplexes forinducing antibodies raised against one or more antigenic proteins in asubject, each heterocomplex comprising (i) a plurality of antigenicproteins, linked to a (ii) carrier protein molecule, characterized inthat less than 40% of the antigenic proteins (i) are covalently linkedto carrier protein molecules (ii).

Most preferably, the antibodies with their production being induced bythe immunogenic product of the invention comprise <<neutralizing>> or<<blocking>> antibodies. A <<neutralizing>> or a <<blocking>> antibodyis defined, according to the invention, as an antibody the binding ofwhich on the native protein blocks the biological activity of such anative protein, which is an important objective being sought by theinvention, when the native protein against which the antibodies areraised has a deleterious biological activity for the organism, withinthe targeted pathological context of an individual to be treated, forexample, when the native protein has an angiogenic activity, animmunosuppressive activity, as well as an allergenic activity, moreparticularly an interleukin-4 production inducing activity.

A <<carrier protein molecule>>, included in the immunogenic product ofthe invention, means any protein or peptide being at least 15 aminoacids long, whatever its amino acid sequence, and which, when partiallycovalently being associated to the molecules of the antigen of interestfor forming protein heterocomplexes making up the immunogenic product ofthe invention, allows for a large number of molecules of the antigen ofinterest to be presented to the B lymphocytes.

According to a first aspect, the carrier protein molecule consists inone protein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising one or more auxiliary T epitopes(“helper”) able to activate auxiliary T lymphocytes (“T helper”) of thehost organism for producing cytokins, including interleukin 2, suchcytokins, in turn, activating and inducing the proliferation of Blymphocytes, which, after maturation, will produce antibodies raisedagainst the antigenic protein (i).

According to a second aspect, a carrier protein molecule consists in oneprotein or one peptide being at least 15 amino acid long, or also anoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”), as described in the above-mentioned first aspect,one or more cytotoxic T epitopes, able to induce a cell immune responsethrough the production of cytotoxic T lymphocytes specific of thecarrier protein molecule, such lymphocytes being able to specificallyrecognize cells expressing on their surface said carrier protein or anypeptide being derived therefrom, in association with class 1Histocompatibility Major Complex (HMC) molecules. If need be, thecarrier protein molecule consists in one oligomer of one protein or onepeptide, further comprising be'sides one or more T helper epitopes, oneor more above defined cytotoxic T epitopes.

According to a third aspect, a carrier protein molecule consists in oneprotein or one peptide being at least 15 amino acid long, as well as oneoligomer of such a peptide, comprising besides one or more auxiliary Tepitopes (“helper”) as defined in the first aspect, one or more Bepitopes, able to induce the production of antibodies by lymphocytesraised against the carrier protein.

In some embodiments, the carrier protein, besides its T helper, used foractivating an antibody response against the antigen of interest, couldalso activate a cytotoxic respone against cells carrier peptides of thecarrier and/or stimulate an antibody response against such a carrierprotein molecule.

The carrier protein molecule could also consist in a homo-oligomer or ahomo-polymer of the native protein, from which it is derived, as well asa homo-oligomer or a homo-polymer of a peptide fragment of the nativeprotein, from which it is derived. The antigenic protein of interestcould also consist in a hetero-oligomer or a hetero-polymer comprising acombination of several distinct peptide fragments initially included inthe native protein from which it is derived.

As used herein, the expression <<antigenic protein>> means any proteinor any peptide being at least 10 amino acid long, including a haptenpeptide, able to be specifically recognized by receptors for theantigens expressed by the B lymphocytes of a host organism, whetherhuman or animal, more particularly a mammal, such antigenic protein,once included in an immunogenic product of the invention, stimulatingthe production of antibodies recognizing said antigenic protein.

It is meant under <<antigenic protein>> any protein comprising one ormore B epitopes of the native antigenic protein against which theproduction of antibodies if being sought. Said antigenic molecule ofinterest could consist in the native protein itself or a proteinderivate of the native protein, such as a peptide fragment of the nativeprotein, as well as any biologically inactivated form of the nativeprotein obtained through chemical, physical treatment or geneticmutation. The antigenic molecule of interest could also consist in ahomo-oligomer or a homo-polymer of the native protein as well as ahomo-oligomer or a homo-polymer of a peptide fragment of the nativeprotein. The antigenic protein of interest could also consist in ahetero-oligomer or a hetero-polymer comprising a combination of severaldistinct peptide fragments initially included in the native protein.

In an immunogenic product according to the invention, advantageously,less than 30% and preferably less than 20% of antigenic proteins (i) arecovalently linked to the carrier protein molecules (ii).

In an immunogenic product according to the invention, advantageously, atleast 1%, and preferably at least 2%, of the antigenic proteins (i) arecovalently linked to the carrier molecules (ii).

It has been shown that an immunogenic product according to theinvention, such as hereinabove defined, is stable in an aqueoussolution. The stability of an immunogenic product of the invention ismore particularly characterized in that said immunogenic product has itsown isoelectric point, distinct from the isoelectric point of at leastone of its protein components, respectively the antigenic protein (i)and the carrier protein molecule (ii), and in that it therefore migratesaccording to a distinct protein strip from at least one of its proteinstrips respectively corresponding to both protein components making itup in isoelectrofocusing trials.

It has also been shown, through immunoblotting trials (<<Westernblot>>), that the immunogenic product of the invention migrates in anelectrophorese gel, under non denaturating conditions, according to asingle protein strip, which illustrates the fact that said immunogenicproduct has the form of a homogeneous population of soluble proteinconstructions.

Moreover, it has been shown that the antigenic protein (i) as well asthe protein molecule (ii) included under the form of proteinheterocomplexes in the immunogenic product of the invention were bothrecognized by antibodies specifically recognizing each of such proteins.Thus, the immunogenic product according to the invention comprises theantigenic protein (i) and the carrier protein molecule (ii) in theirnative structure. Such a technical feature of the immunogenic productaccording to the invention is particularly advantageous for inducing animmune response against native antigens, i.e. an efficient and trulyprotective immune response of the host organism. It has been moreparticularly shown that an immunogenic product according to theinvention induces, in the host organism to which it is administered, theinduction of a strong efficient humoral response against nativeantigens, associated to the production of neutralizing or blockingantibodies, towards the deleterious biological activity of such nativeantigens.

It has been shown according to the invention, with various antigens ofinterest, that the humoral immune response obtained using an immunogenicproduct such as defined hereinabove, was 10 to 1000 times higher thanthe humoral immune response obtained with the administration of aconventional covalent conjugate between the antigen of interest and thecarrier protein molecule.

Preferably, in an immunogenic heterocomplex included in the immunogenicproduct of the invention, the plurality of antigenic proteins (i) ismade up of a plurality of specimens of a single antigenic protein.

Thus, according to a most preferred embodiment, the immunogenic productof the invention is implemented for obtaining specific antibodies raisedagainst a single antigen of interest.

It has also been shown according to the invention that an immunogenicproduct comprising immunogenic heterocomplexes such as hereinabovedefined, is particularly well adapted to the immunization of anindividual, through the production of antibodies, against a <<selfantigen>> of interest, i.e. against a protein being naturally producedby said individual, for which there exists a tolerance of the immunesystem, in particular an at least partial deletion of auxiliarylymphocyte T clones (T helper cells) specifically recognizing saidantigen.

In other words, the presentation of the <<self>> antigen to the cells ofsaid individual's immune system, under the form of an immunogenicproduct comprising immunogenic heterocomplexes of the invention, allowsto <<break>> the tolerance of the individual's immune system towardssuch an antigen. Without wishing to be bound to any theory, theApplicant believes that the opportunity to obtain a high level ofantibody response against a <<self>> antibody is due to the presencewithin the heterocomplex of numerous epitopes of the <<auxiliary T>>type (or T helper) carried by the carrier protein molecule, activatingthe auxiliary T lymphocytes, and the various cytokins produced by theactivated auxiliary T lymphocytes, including IL-2, allows to promotesome activation of the B cells to <<self>> antigens present in thelatent state within the organism, and to thereby break the immunetolerance of B cells to <<self>> antigens.

Thus, according to a preferred embodiment, the immunogenic product ofthe invention is characterized in that the antigenic proteins (i)consists in a plurality of specimens of a protein being normallyrecognized as a self protein by the cells of said subject's immunesystem.

As the major proportion, more than 60%, of associations between theantigen of interest and the carrier protein molecule, occurs through noncovalent interactions, there exists no other theoretical limitation inthe number of molecules of the antigen of interest associated with asingle carrier protein molecule, than the steric availability of themolecules of the antigen of interest to such a carrier molecule. Inparticular, the number of molecules of the antigen of interestassociated to a single carrier protein molecule is not limited by thenumber of chemically reactive functions carried by the carrier moleculeallowing for creating covalent links with a plurality of molecules ofthe antigen of interest. Consequently, the only physical limitationseems to be the number of sites of the carrier protein molecule (ii)available to the antigenic protein (i).

For the same reasons, the size of the antigen of interest to beassociated to the carrier protein molecule is not either strictlylimited, the antigen of interest consequently being able to consist infull proteins of at least 10 kDa, such as the various cytokins, as IL-4,IL-10, VEGF as well as the alpha interferon.

Moreover, even for the antigens of interest consisting in full proteinswith a molecular mass higher than 10 kDa, an immunogenic heterocomplexof the invention can comprise an association of several antigens ofinterest on a single carrier molecule, if the size of the carriermolecule makes it possible.

When the carrier protein molecule has a small size, for example a sizelower than 10 kDa, or even lower than 5 kDa, the Applicant believes,without wishing to be bound to any theory, that the partially covalentassociations between the antigen of interest and said carrier protein,forming the protein heterocomplexes included in the immunogenic productof the invention, allow for such a conformation of heterocomplexes thatboth the antigen of interest and the carrier protein molecule areavailable to receptors of the immune system cells.

This is even an additional technical advantage provided to theimmunogenic product comprising heterocomplexes such as hereinabovedefined, as the presentation to B lymphocytes of a plurality ofspecimens on one single carrier molecule, included in the heterocomplex,enhances the <<capping>> phenomenon through <<cross-linking>> ofreceptors of the B cell recognizing the antigen, contributing to theactivation of the B cell receiving, in addition, activation signalscoming from cytokins produced by the activated auxiliary T lymphocytesactivated by means of auxiliary T epitopes carried by carrier proteinmolecule.

Thus, according to a most preferred embodiment of the immunogenicproduct, the latter comprises 5 to 50 antigenic proteins (i) for onecarrier protein molecule (ii), preferably 20 to 40 antigenic proteins(i) for one carrier protein molecule (ii).

The number of molecules of the antigen of interest on one single carrierprotein molecule respectively depends on the size of the carriermolecule and on the size of the molecule of the antigen of interest. Thebigger the carrier molecule is and offers a large association surfacewith the antigen of interest, the more the immunogenic heterocomplexwill comprise, for one single of the carrier molecules it contains, ahigher number of specimens of the molecule of the antigen of interest.Similarly, the more reduced the size of the molecule of the antigen ofinterest is, the larger the number of specimens will be of the moleculeof the antigen of interest on the same carrier molecule.

By way of illustration, it has been shown according to the inventionthat when the carrier protein molecule is KLH, 20 to 40 molecules ofIL-4, IL-10, alpha interferon or VEGF are associated to each carriermolecule.

It has been shown that the solubility of the immunogenic product in anaqueous solution varies with the modification of the balances masteringthe molecular interactions within heterocomplexes, more particularly theelectrochemical balances depending on the so-called <<weak>> (noncovalent) links as well as the respective concentrations in antigenicproteins and the carrier protein molecule, as well as with theconditions of ionic strength, pH and temperature.

Preferably, the covalent bonds between one or more antigenic proteins(i) and the carrier protein molecule (ii) occur by means of abifunctional bonding chemical agent.

Such a chemical agent could be cyanogen bromide, glutaraldehyde,carbodiimide or succinic anhydride.

As for carbodimides, the following compounds could be used:1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) and1-ethyl-3-(4-azonia-4,4-limethylpentyl)carbodiimide,1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide,(1-ethyl-3-(3-dimethyaminopropyl carbodiimide (EDC) and1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

As homo-bifunctional coupling agents, the following compounds could beused:

N-hydroxysuccinimide, dithiobis(succinimidylpropionate) esters,disuccinimidyl suberate, and disuccinimidyl tartrate; bifunctionalimidoesters dimethyl adipimidate, dimethyl pimelimidate, and dimethylsuberimidate;

reagents with a sulphydryl,1,4-di-[3′-(2′-pyridyledithio)propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane;

bifunctional halides of the aryl type and4,4′-difluoro-3,3′-dinitrophenylsulfone;

SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate);

SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate);

SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate);

GMBS (N-(.gamma.-maleimidobutyryloxy)succinimide ester);

MPBH (4-(4-N-maleimidophenyl)hydrazide butyric acid);

M2C2H (4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide);

SMPT(succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene);and

SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

Preferably, the bonding chemical agent to be used comprises at least tworeactive aldehyde functions.

Most preferably, the bonding chemical agent is glutaraldehyde.

After the product comprising protein heterocomplexes has been formedthrough a coupling of carrier protein molecules with antigenic proteinswith the use of the bonding chemical agent, the resulting product couldbe stabilized by means of a protein stabilizing agent, such asformaldehyde, able to create intrachain bonds.

The immunogenic products comprising immunogenic heterocomplexes of theinvention have the form of microparticles soluble in solution, inparticular in an aqueous solution, their average size varying dependingon (i) the size of the carrier protein molecule, (ii) the size and thenumber of antigenic proteins associated to one single carrier proteinmolecule and (iii) the number of carrier molecules associated to theantigenic proteins present in a heterocomplex particle.

It has been found that the heterocomplex microparticles described in theexamples have an average size ranging from 100 nm to 300 nm.

Most preferably, an immunogenic product comprising immunogenic proteinheterocomplexes of the invention exclusively comprises carrier moleculesassociated to antigenic proteins, with the exclusion of any othermaterial. More particularly, a heterocomplex of the invention does notcomprise any other polymeric, proteinaceous or non proteinaceousmaterial, other than the carrier and antigenic proteins characterizingit.

Recently, ZAGURY D et al. (2001, Proc. Nail. Acad. Sci. USA.98(14):8024-8029), in a bibliographical study, suggested to induce ananti-cytokin immunity in patients in order to counteract the abnormalproduction in such pathologies of some cytokins, including interleukins,lymphokins, monokins, interferons, physiologically acting in thetissues, locally as a factor of programmed cell proliferation,differentiation, or death.

The above-mentioned authors state that the strategies of vaccine therapywere, until now, exclusively focused on the antigenic aggressor, whetherit is a micro-organism, a cell or an allergen, but never attempted tofight the deregulation of cytokins induced under the effect of theaggressor. Said authors suggest an anti-cytokin vaccination as a priorstep to a conventional vaccination having as an aim to neutralize orblock the immunotoxic effect of the stroma, and to allow for the normaloccurrence of the immune reaction adapted towards the antigenicaggressor.

Moreover, the Applicant's prior work, mentioned in the InternationalApplication published under WO 00/03732, showed that in the case of ATLleukemia, the neck of the uterus cancer and the Kaposi sarcoma,respectively, three proteins are involved in a local immunosuppressionat the level of tumors or HIV1 infected cells:

-   -   the HTLV1 virus Tax protein,    -   the papillomavirus E7 protein, and    -   the V1H-1 virus Tat protein.

The Applicant also stated that some of such immunosuppressive proteins,such as the HIV1 Tat protein and the HPV E7 protein (Strains 16 and 18)also have activating effects on vascular endothelial cells.

They therefore suggested developing anti-cancer or anti-viral vaccinescomprising a detoxicated immunogenic compound derivate of a proteincoming from cancer cells, from virus infected cells or stroma immunecells, initially immunosuppressive and/or angiogenic with a localaction, as, for example, a protein derived from the H1V1 virus Tatprotein, the HTLV1 virus Tax protein, papillomavirus E7 protein as wellas a mannan-depending lectin under an inactivated form.

Now, it has been shown according to the invention that the immunogenicproduct comprising immunogenic heterocomplexes such as hereinabovedefined allows for the induction of a strong antibody response againstthe various above-mentioned deleterious antigenic molecules.

According to a first aspect, in the immunogenic heterocomplex of theinvention, the antigenic protein(s) (i) consist(s) in cytokins naturallyproduced by said subject.

Preferably, the antigenic protein(s) (i) is/are selected frominterleukin-4, alpha interferon, gamma interferon, VEGF, interleukin-10,alpha TNF, beta TGF, interleukin-5 and interleukin-6.

According to a second aspect, the antigenic protein(s) (i) making up animmunogenic heterocomplex of the invention is/are immunosuppressive orangiogenic proteins, or proteins derived from immunosuppressive orangiogenic proteins.

Preferably, the antigenic protein(s) (i) is/are selected amongst apapillomavirus E7 protein, the VIH 1 virus Tat protein, the HTLV 1 orHTLV 2 virus Tax protein, and the self p53 protein.

According to a third aspect, the antigenic protein(s) (i) making up animmunogenic heterocomplex according to the invention is/are proteinsbeing toxic at a low dose to man or to a non human mammal. These aremore particularly various proteins being lethal to man at a dose lowerthan 1 mg, lower than 100 μg, lower than 10 μg, even lower than 1 μg.These are predominantly toxic proteins able to be used for manufacturingso-called <<biological>> weapons, such as ricin, botulic toxins,staphylococcus enterotoxins, as well as an anthrax toxic protein (EF,LF, PA).

The carrier protein molecule (ii) included in an immunogenic proteinheterocomplex of the invention could be a carrier moleculeconventionally used in immunology, such as KLH, ovalbumin, bovine serumalbumin (BSA), toxoid tetanos, B cholera toxin, etc.

Moreover, in an immunogenic product of the invention, the proteincarrier molecule could be selected so as to induce or stimulate, besidesthe production of T helper lymphocytes, a cytotoxic and/or humoralimmune response against itself, and its counterpart of native protein inthe host organism, respectively through the activation of cytotoxic Tlymphocytes and of B lymphocytes specific to such a carrier molecule.

Such a particular embodiment of an immunogenic product of the inventionis particularly useful when there is simultaneously sought an efficientantibody response against an immunosuppressive or angiogenic deleteriousprotein, more particularly, for producing neutralizing or blockingantibodies, and a cell immune response generated by cytotoxic Tlymphocytes raised against cells having at their surface the nativeantigen associated to Major Histocompatibility Complex (MHC) class Imolecules, for example, an antigen of a pathogen, such as the VIH1 virusor a papillomavirus, or an antigen specifically expressed in cancercells such as CEA, p53, Di12, etc.

Thus, according to this particular embodiment, the immunogenic productof the invention is characterized in that the carrier protein molecule(ii) is an immunogenic protein inducing, besides the production of Thelper lymphocytes, the production of cytotoxic T lymphocytes raisedagainst cells having at their surface said carrier protein molecule, orany peptide being derived therefrom, in association with MajorHistocompatibility Complex (MHC) class I molecules and/or the productionof antibodies by B lymphocytes raised against the carrier protein.

Thus, immunogenic products comprising immunogenic heterocomplexes of theinvention are efficient immunologic means for the active therapeuticvaccination of an individual, whether a human mammal or a non humanmammal, against a large variety of pathologies.

Illustrative examples of such immunogenic heterocomplex compositionscontained in an immunogenic product according to the invention forpreventing or treating, through an active therapeutic vaccination,various pathologies are mentioned hereinafter.

a) For Preventing or Treating AIDS:

Carrier protein molecule (ii): gp 120, gp 160, p24, p17, nef or Tatproteins of HIV1 virus, detoxicated or stabilized if required,immunogenic fragments of such proteins as well as an immunogenic proteinbeing derived therefrom (Zagury et al., 1998).

The mimotope gp 120 protein could also be used as described by Fouts etal. (2000) and by Fouts et al. (2002).

Antigenic protein (i): Tat, IFNα, IL10 and TGFβ proteins, detoxicated ifrequired, immunogenic fragments of such proteins, or an immunogenicprotein being derived therefrom.

b) For Preventing or Treating the Neck of Uterus Cancer:

Carrier protein molecule (ii): papillomavirus L1, L2 and E7 proteins,preferably a papillomavirus from strain 16 or 18, detoxicated orstabilized if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom (Le Buanec et al.,1999).

Antigenic protein (i): E7, IFNα, IL10, TGFβ, TNFα and VEGF proteins,detoxicated or stabilized if required, immunogenic fragments of suchproteins as well as an immunogenic protein being derived therefrom.

c) For Preventing or Treating ATL Leukemia Induced by the HTLV1 or 2Viruses:

Carrier protein molecule (ii): gp61 and HTLV1 or 2 virus Tax proteins,detoxicated if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom (Cowan et al., 1997;Mori et al., 1996).

Antigenic protein (i): Tax, IL10, IFNα or TGFβ, TNFα, VEGF proteins,detoxicated if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom.

d) For Preventing or Treating Colon Cancer:

Carrier protein molecule (ii): CEA and p53 proteins, detoxicated ifrequired, immunogenic fragments of such proteins as well as animmunogenic protein being derived therefrom (Zusman et al., (1996)).

Antigenic protein (i): IFNα, TGFβ, IL10, FasL and VEGF proteins,detoxicated if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom.

e) For Preventing or Treating Breast Cancer:

Carrier protein molecule (ii): Di12 protein, immunogenic fragments ofsuch a protein as well as an immunogenic protein being derived therefrom(Yoshiji et al., 1996).

Antigenic protein (i): IFNα, TGFβ, IL10, FasL and VEGF proteins,detoxicated if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom.

f) For Preventing or Treating Pancreas Cancer:

Carrier protein molecule (ii): CaSm protein, detoxicated if required,immunogenic fragments of such proteins as well as an immunogenic proteinbeing derived therefrom.

Antigenic protein (i): VEGF and TNFα proteins, detoxicated or stabilizedif required, immunogenic fragments of such proteins as well as animmunogenic protein being derived therefrom.

g) For Preventing or Treating Prostate Cancer:

Carrier protein molecule (i): OSA and ETS2 proteins, detoxicated orstabilized if required, immunogenic fragments of such proteins as wellas an immunogenic protein being derived therefrom. (Sementchenko V I etal., 1998).

Antigenic protein (i): IL6 and TGFβ proteins, detoxicated or stabilizedif required, immunogenic fragments of such proteins as well as animmunogenic protein being derived therefrom (Adler et al., 1999).

h) For Preventing or Treating Some Allergies:

Carrier protein molecule (ii): it is selected amongst molecularallogens, such as Bet v 1 (birch-tree pollen), Der p 1 (acarid) and Feld 1 (cat) proteins, their immunogenic peptide fragments as well as animmunogenic protein being derived therefrom. The Bet v 1 antigen isdescribed more particularly by Ferreira et al. (1993), the Der p 1antigen is described, in particular, by Tovey et al. (1981) and the Feld 1 antigen is described, more particularly by Morgensterm et al. (1991)

Antigenic protein (i): it induces the production of neutralizing orblocking antibodies raised against the IL4 cytokin factor, being mainlyproduced by T lymphocytes of Th2 type, orienting the humoral immuneresponse towards the production of IgE isotype antibodies. According toanother embodiment, the antigenic protein (i) induces the production ofneutralizing or blocking antibodies against the IL5 cytokin factor,being mainly produced by T lymphocytes of Th2 type.

According still another embodiment, preventing allergy could occur bymeans of an immunogenic product inducing an antibody response againstthe main basophil granulation effector, i.e. IgE isotype antibodies. Forthis purpose, the invention provides an immunogenic product comprising(i) an IgE isotype human antibodiy and (ii) the VIH1 inactivated Tatprotein.

i) For the Prevention Against Lethal Proteins Used in Biological Weapons

Also, an immunogenic product according to the invention could be usedfor immunizing individuals against numerous toxic products used, inparticular, in chemical and biological weapons, as for example, ricin.

Amongst the most toxic proteins against which an immunization, mainlythrough the production of antibodies, is being sought, botulic toxins,ricin, staphylococcus enterotoxins, Clostridium perfringens toxins andanthrax toxic proteins.

Generally speaking, for producing an immunogenic product according tothe invention, wherein the antigenic protein (i) is a highly toxicprotein of the above-mentioned type, a previously detoxicated protein isused under the form of a toxoid. For detoxicating the protein, beforeits use for producing an immunogenic product according to the invention,various methods could be used, and preferably one of the followingmethods consisting in:

a) treating the native toxic protein by glutaraldehyde;

b) treating the native toxic protein through the combined action offormol and glutaraldehyde; or

c) if need be, through chemical modification of His and Tyr groups bymeans of appropriate reagents, for example, through carboxymethylationof such amino acid residues.

For preventing the lethal action of toxins originating from Bacillusanthracis, as proteins, antigenic proteins (i), a detoxicated proteinoriginating from an anthrax protein selected amongst EF (<<EdemaFactor>>), LF (<<Lethal Factor>>) and PA (<<Protective Antigen>>)proteins are used preferably.

For preventing the lethal actions of proteins originating fromClostridium perfringens, as the antigenic protein (i), a detoxicatedprotein originating from the Epsilon toxin of Clostridium perfringensare preferably used.

For preventing the lethal action of toxins originating form Clostridiumbotulinum, as antigenic proteins (i), a detoxicated protein originatingfrom a botulic toxin selected amongst A, B, C, D, E, F and G toxinsbeing naturally synthetized in the form a single 150 kDa polypeptidechain as well as the H_(c) fragment of such botulinic toxins, saidfragment H_(c) having a molecular mass of approximately 50 kDa, arepreferably used.

For producing inactivated botulic toxins, the man of the art could usetechniques known per se, more particularly those used for preparing theanterior vaccine compositions, such as those described by Fiock et al.or by Siegel et al. (Fiock, M. A., Cardella, M. A., Gearinger, N. F., J.Immunol., 1963, 90, 697-702; Siegel, L. S., J. Clin. Microbiol., 1988,26, 2351-2356).

For preventing the lethal action of toxins originating from ricin seed(Ricinus communis), as the antigenic protein (i), a detoxicated proteinoriginating from the ricin toxin, preferably the β fragment of ricin, ispreferably used.

Thus, the invention also provides an immunogenic product comprising (i)the β fragment of ricin and (ii) the KLH protein.

For purifying ricin, the man of the art could use any known technique,such as those described by Osborne et al., Kabat et al. or Kunnitz etal. (Osborne, T. B., Mendel, L B. and Harris, J. F.: Amer. J. Physiol.,1905, 14, 259-269; Kabat, E. A. Heidelberger, M. and Bezer, A. E.: J.Biol. Chem., 1947, 168, 629-; Kunnitz, M. and McDonald, M.: J. Gen.Physiol., 1948, 22, 25- Moulé, Y.: Bull. Soc. Chim. Biol., 1951, 33,1461-1467). He could also make use of the affinity chromatographypurification techniques described by Tomila et al., Nicolson et al. orOlsnes et al. (Tomila, M., Kurokawa, T., Onozaki, K. et a/.:Experientia, 1972, 28, 84-85; Nicolson, G. L. and Blaustein, J.: J.Biochim. Biophys. Acta, 1972, 266, 543-547; Olsnes, S., Salvedt, E. andPihl, A.: J. Biol. Chem., 1974, 249, 803-810). The ricin A and B chainscould be purified as described by Hedge et al. (Hedge, R. and Podder, S.K.,: Eur. Biochem., 1998, 254, 596-601).

For preventing the lethal action of toxins originating fromstaphylococcus and more particularly from Staphylococcus aureus, as theantigenic protein (i), a detoxicated protein originating from a toxinselected amongst SEA (<<Staphylococcal Enterotoxin A>>), SEB(<<Staphylococcal Enterotoxin B>>), SEC (<<Staphylococcal EnterotoxinC>>), SED (<<Staphylococcal Enterotoxin D>>), SEE (<<StaphylococcalEnterotoxin E>>), SEG (<<Staphylococcal Enterotoxin G>>), SEH(<<Staphylococcal Enterotoxin H>>), SEI (<<Staphylococcal EnterotoxinI>>) and TSST-1 (<<Toxic Shock Syndrome Toxin-1>>) is preferably used.

The above listed enterotoxins could be prepared by the man of the art bymeans of techniques described in the listed work below, relating to thedescription of each of such toxins.

SEA is synthesized in the form of a precursor enterotoxin with 257 aminoacids (Huang, I. Y., Hughes, J. L, Bergdoll, M. S. and Schantz, E. J. JBiol. Chem. 1987, 262, 7006-7013). The mature toxin with a molecularmass equal to 27,100 Da derives from the precursor toxin through theloss of a N-terminal hydrophobic leader sequence with 24 amino acidresidues (Betley, M. J. and Mekalanos, J. J. J Bacteriol., 1998, 170.34-41). SEA exists under 3 different isoforms through their IP.

The SEB precursor protein comprises 267 amino acids (Mr=31,400 Da) witha N-terminal signal peptide with 27 amino acids. Its binding site to thereceptor of T cells (<<T-Cell Receptor>> or <<TCR>>) encompasses theshallow cavity, whereas the class II MHC molecule is fixed on anadjacent site (Kappler, J. W., Herman, A., Clements, J. and Marrack, P.:J. Exp. Med., 1992, 175, 387-396; Papageorgiu, A. C., Trauter, H. S. andAcharya, K. R. J Mol. Biol., 1998, 277, 61-79; Soos, J. M. and Johnson,H. M. Biochem. Biophys. Res. Commun., 1994, 201, 596-602).

SEC possesses 3 antigenically distinct sub-types: SEC 1, SEC 2 et SEC 3.The precursor proteins contains 267 amino acid residues (Houde, C. J.,Hackett, S. P. and Bohach, G. A. Mol. Gen. Genet., 1990, 220, 329-333)with a signal peptide with 27 amino acid residues (Bohach, G. A. andSchlievert, P. M.: Infect. Immun., 1989, 57, 2243-2252).

SED is made up of 258 amino acid residues with a signal peptide of 30amino acid residues. Its three-dimension structure is similar to thestructure of other bacterial superantigens.

SEE having a 26,000 Da molecular mass have 81% of AA sequence homologywith SEA.

SEG is made up of 233 amino acid residues (Munson, S. H., Tremaine, M.T., Betley, M. J. and Welch, R. A.: Infect. Immun., 1998, 66,3337-3348).

SEH has a 27,300 Da molecular mass (Su, Y. C. and Wong, A. C.: Apll.Environ. Microbiol., 1995, 61, 1438-1443). It does not have any crossedimmunologic reaction with other enterotoxins.

SEI has a sequence comprising 218 amino acid residues. This is the toxinwith the lowest homology level with other enterotoxins

SEJ made up of 269 amino acid residues has a high AA sequence homologywith SEA, SEE and SED (64-66%).

Preferably, an immunogenic product according to the invention comprises,in combination, several antigenic proteins (i) each derived from anabove-mentioned toxic protein, for example, 2, 3, 4 or 5 antigenicproteins (i) each derived from an above listed toxic protein.

For example, an immunogenic product according to the invention forpreparing a vaccine composition intended for preventing the toxicity ofstaphylococcus enterotoxins preferably comprises 2, 3, 4 or 5 antigenicproteins (i) each derived from a staphylococcus enterotoxin.

According to a particular embodiment of an immunogenic product accordingto the invention, wherein the antigenic protein(s) (i) is/are derivedfrom highly toxic proteins for man, the carrier protein is the KLHprotein.

Thus, according to a first particular aspect of an immunogenic productof the invention, wherein the carrier protein molecule both induces theproduction of auxiliary T lymphocytes (<<T helper>>), of cytotoxic Tlymphocytes and of B lymphocytes specific to the carrier proteinmolecule, said carrier protein molecule (ii) is selected amongst thepapillomavirus L1, L2, and E7 proteins.

Thus, according to a second particular aspect of an immunogenic productof the invention, wherein the carrier protein molecule induces, inaddition to the production of auxiliary T lymphocytes (<<T helper>>),the differentiation of cytotoxic T lymphocytes and of B lymphocytesspecific to the carrier protein molecule, said carrier protein molecule(ii) is selected amongst the HIV1 virus gp160, p24, p17, Nef and Tatpoteins.

Thus, according to a third particular aspect of an immunogenicheterocomplex of the invention, wherein the carrier protein moleculeboth induces the production of auxiliary T lymphocytes (<<T helper>>),of cytotoxic T lymphocytes and of B lymphocytes specific to the carrierprotein molecule, said carrier protein molecule (ii) is selected amongstCEA, p53, Di12, CaSm, OSA and ETS2 proteins.

According to a fourth particular aspect of an immunogenic product of theinvention, wherein the carrier protein molecule induces, in addition tothe differentiation of auxiliary T lymphocytes (<<T helper>>), theproduction of antibodies raised against the carrier protein molecule,said carrier protein molecule (ii) is selected amongst Bet v 1, Der p 1and Fel d 1 proteins.

In an immunogenic product according to the invention, the immunogenicprotein heterocomplexes are selected amongst the followingheterocomplexes, where the antigenic proteins (i), on the one hand, andthe protein carrier molecule (ii), on the other hand, are respectively:

a) (i) IL-4 and (ii) KLH;

b) (i) alpha interferon and (ii) KLH;

c) (i) VEGF and (ii) KLH;

d) (i) IL-10 and (ii) KLH;

e) (i) alpha interferon and (ii) gp 160 of VIH1;

f) (i) IL-4 and (ii) the Bet v 1 allergenic antigen; and

g) (i) VEGF and (ii) the papillomavirus E7 protein;

h) (i) the inactivated VIH1 Tat protein and (ii) the VIH1 gp 120protein;

i) i) an IgE isotype human antibody and (ii) the inactivated VIH1 Tat;

j) (i) the ricin β fragment and (ii) KLH.

Method for Preparing an Immunogenic Product Comprising ImmunogenicProtein Heterocomplexes of the Invention

Another object of the invention is also a method for preparing animmunogenic product comprising the hereinabove defined immunogenicheterocomplexes, characterized in that it comprises the following stepsof:

a) incubating the antigenic proteins (i) and the carrier molecule (ii)in a molar ratio (i):(ii) ranging from 10:1 to 50:1 in the presence of abinding chemical agent;

b) collecting the immunogenic product comprising immunogenicheterocomplexes being prepared in step a).

Preferably, the binding chemical agent is glutaraldehyde.

Most preferably, the method is further characterized in that step a) isfollowed by a stabilizing step of the product comprising the immunogenicheterocomplexes by formaldehyde, prior to step b) for recovering theheterocomplexes.

Preferably, when glutaraldehyde is used as the binding chemical agent,it is present in the coupling reaction medium in a final concentrationranging between 0.002M and 0.03M, advantageously between 0.02M and0.03M, preferably in a final concentration of 0.026M.

The coupling reaction with glutaraldehyde advantageously occurs for 20minutes to 60 minutes, preferably 30 minutes, at a temperature rangingfrom 20 to 25° C.

After the coupling step, the excess glutaraldehyde is removed, forexample, through dialysis by means of a dialysis membrane with a 3 kDacutoff threshold. The dialysis step advantageously occurs at 4° C. in abuffer adjusted to pH 7.6.

For stabilizing the product comprising the protein heterocomplexes asprepared in step a), said product could be treated in solution by theformaldehyde, for example, by formaldehyde in a final concentration of 3mM. The stabilization reaction is advantageously performed for 12 to 48hours, preferably between 20 and 30 hours, and most preferably, for 24hours. The stabilization reaction using the formaldehyde isadvantageously stopped through the addition of glycine, preferably in a0.1M concentration, for 1 hour and at a temperature ranging from 20 to25° C.

The Compositions Comprising an Immunogenic Product ComprisingImmunogenic Protein Heterocomplexes of the Invention

Another object of the present invention is also to provide a compositioncomprising an immunogenic product such as hereinabove defined.

The invention also relates to a pharmaceutical composition comprising aprotein immunogenic product such as hereinabove defined.

Another object of the invention is an immunogenic compositioncharacterized in that it comprises, as the active ingredient, animmunogenic product as hereinabove defined, in association with one ormore physiologically compatible excipients.

It also relates to a vaccine composition characterized in that itcomprises, as the active ingredient, an immunogenic product ashereinabove defined, in association with one or more physiologicallycompatible excipients.

Depending on the target objectives, systemic adjuvants or mucosaladjuvants are being used. For example, a mucosal adjuvant is preferablyused for preventing the epithelial tissue cancers and preferablysystemic adjuvants are used for preventing or treating virus infectionssuch as HIV1 and HTLV1 as well as for preventing or treating allergies.

Amongst systemic adjuvants, those of the IFA type are preferably used(Incomplete Freund's Adjuvant), as well as calcium phosphate or aluminahydroxide.

Amongst mucosal adjuvants, those preferably used are like B chloratoxin(CTB) or a mutant of the LT toxin (LTμ).

According to a particular aspect, an immunogenic composition accordingto the invention also comprises one or more immuno-stimulating agents,in combination with an immunity adjuvant, such as for example, the CpGimmuno-stimulating agent well known in the state of the art.

It has indeed been shown according to the invention that the use of theCpG adjuvant, and more particularly the CpG adjuvant wherein theintra-chain bonds between nucleotides consist in phosphorothioate bondsto stimulate the simultaneous production of IgG and IgA isotypeantibodies, after a systemic administration.

It also relates to a mucosal or systemic vaccine, characterized in thatit comprises, as the active ingredient, an immunogenic product such ashereinabove defined, in association with one or more excipients,including physiologically compatible adjuvants.

The immunogenic compositions or the vaccines according to the presentinvention are useful for example in the treatment, both curative andpreventive, of cancers, more particularly, of cancers induced by virusessuch, as for example, the ATL (Acute T cell leukemia) caused by theHTLV1, or the neck of uterus cancer caused by the papillomavirus, aswell as the Burkitt lymphoma as well as the Kaposi sarcoma caused by theviruses from the herpes family, respectively the Epstein-Barr (EBV) andthe HHV8 as well as in treatment of AIDS or for preventing or treatingallergic reactions.

The immunogenic products according to the invention could be used asfollows.

To a patient, is administered, under a form adapted to the systemic ormucosal administration, an immunogenic product comprising immunogenicprotein heterocomplexes according to the present invention, for example,intranasally, in a sufficient amount to be therapeutically efficient, toa subject in need of such a treatment. The dose to be administered couldrange for example from 10 to 1000 μg intranasally, once a week for 2months and then, given the transitory character of the antibody responsedirected against the antigen of interest, periodically depending on theserum antibody rate, for example, once every 2 to 6 months.

Two or more different immunogenic products could be administered in onesingle preparation for inducing neutralizing antibodies in all thedeleterious functional sites should one single molecule not carry allthe active sites of the overproduced toxin or cytokin which is to beneutralized.

As for drugs, the immunogenic products of the invention could beincorporated into pharmaceutical compositions adapted for anadministration through the systemic route or an administration throughthe mucosal route, including the oromucosal route, more particularly,the intranasal route, the oral route and the vaginal route. Theadministration could be performed in one single dose or a dose repeatedonce or several times after some time interval.

This is why the present application has also as an object apharmaceutical, curative, or preventive composition, characterized inthat it comprises as an active ingredient, one or more immunogenicproducts such as hereinabove defined. The immunogenic product could bepackaged alone or mixed with an excipient or a mixture ofpharmaceutically acceptable excipients such as an adjuvant. Amongst theexcipients adapted for the intranasal or oral route, are particularly tobe selected the capryl caproyl macrogol glycerides as Labrasol® from theGATTEFOSSE corporation or alumina hydroxide (Alhydragel, Superfos,Denmark).

For the oral administration according to the invention, the activeingredient will be associated to a mucosal immunity adjuvant, such as aCT, LT or CTB mutant.

Galenic forms are particularly well suited, as described by Boyaka etal. <<Strategies for mucosal vaccine development>> in Am. J. Trop. Med.Hyg. 60(4), 1999, pages 35-45. Are also to be mentioned gastroresistant, more particularly bioadhesive microgranules, such asdescribed by Rojas et al. in Pharmaceutical Research, vol. 16, n^(o) 2,1999, page 255.

Under the particular implemneting conditions, an above-mentioned vaccinepharmaceutical composition will be selected, characterized in that itcomprises a mucosal immunity adjuvant, such a CT mutant (cholera toxin)or a LT mutant E. coli labile enterotoxin).

Under other particular implementing conditions, a vaccine pharmaceuticalcomposition will be selected, characterized in that it contains anadjuvant absorbing the active ingredient, such as alumina hydroxide orgold particles.

Another object of the present invention is a method for preparing acomposition as described hereinabove, characterized in that are mixed,using methods known per se, the active immunogenic product(s) with theacceptable excipents, including pharmaceutical acceptable ones and ifneed be, with a systemic or mucosal immunity adjuvant.

Under preferred implementing conditions of the above-mentioned method,bioadhesive and gastroresistant microgranules are prepared for thedigestive route containing the immunogenic active ingredients and, ifneed be, the adjuvants.

The present invention is further illustrated by the following examples.

EXAMPLES

Examples of Heterocomplex Preparations

Exemple 1 Preparation of Murine KLH-VEGF Heterocomplex

0.58 mg of KLH protein is dissolved in 0.5 ml of 10 mM phosphate buffer,pH 8.5. To this solution is added 1 mg of murine VEGF dissolved in 1 mlof the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature.

The mixture is finally dialyzed under the same conditions as thepreviously performed dialysis.

Example 2 Human KLH-VEGF Heterocomplex

Such a heterocomplex is the active ingredient of a vaccine able tomainly induce in the vaccinee the production of antibodies neutralizingthe human VEGF.

0.58 mg of KLH protein is dissolved in 0.5 ml of 10 mM phosphate buffer,pH 8.5. To this solution is added 1 mg of human VEGF dissolved in 1 mlof the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 3 Preparation of Murine KLH-IL4 Heterocomplex

0.841 mg of KLH protein is dissolved in 0.8 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of murine IL4 dissolvedin 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 4 Preparation of a Human KLH-IL4 Heterocomplex

Such a heterocomplex is the active ingredient of a vaccine able tomainly induce in the vaccinee the production of antibodies neutralizingthe human IL4.

1 mg of KLH protein is dissolved in 1 ml of 10 mM phosphate buffer, pH8.5. To this solution is added 1 mg of murine IL4 protein dissolved in 1ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 24 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 5 Preparation of a KLH-IFNα Complex

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccinee the production of antibodies neutralizing thehuman IFNα.

0.625 mg of KLH protein is dissolved in 0.6 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of human IFNα proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses, each performed in a dialysis tube with a cutoff thresholdbeing 3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 6 Preparation of a gp160-IFNα Complex

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccinee the production of antibodies neutralizing boththe gp160 structure protein of the HIV-1 virus and the immunosuppressiveIFNα cytokin protein. Moreover, such a heterocomplex should be able toinduce a cell reaction (chemiokins, auxiliary T, CTL) raised against theinfected cells expressing the gp160.

0.380 mg of gp160 protein is dissolved in 0.380 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of human IFNα proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 2 hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1 M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 7 Preparation of a gp160-Toxoid Tat Heterocomplex (The TatProtein is Biochemically Inactivated)

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccinee the production of antibodies neutralizing boththe gp160 structure protein of the HIV-1 virus and the extracellular Tatprotein of VIH-1. Moreover, such a complex should be able to induce acell reaction (chemiokins, auxiliary T, CTL) raised against the infectedcells expressing the gp160.

0.550 mg of gp120 protein is dissolved in 0.550 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of toxoid Tat proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

Then the reaction is blocked by the addition of final 0.1 M glycine for1 hour at room temperature. The excess glycine is then removed by 3successive 2 hour dialyses each in a dialysis tube with a 3 kDa cutoffthreshold, at 4° C. against 200 ml of phosphate buffer, pH 7.6, 10 mM.

Example 8 Preparation of a gp160-GM Tat Heterocomplex (The Tat Proteinis Genetically Inactivated)

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccinee the production of antibodies neutralizing boththe gp160 structure protein of the HIV-1 virus and the Tat proteinregulating the VIH-1. Moreover, such a heterocomplex should be able toinduce a cell reaction (chemiokins, auxiliary T, CTL) raised against theinfected cells expressing the gp160.

0.550 mg of gp160 protein is dissolved in 0.550 ml of 10 mM phosphatebuffer, pH 8.5. To this solution is added 1 mg of GM Tat proteindissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 30 minutes at room temperature.

Then the reaction is blocked by the addition of final 0.1M glycine for 1hour at room temperature. The excess glycine is then removed by 3successive 2 hour dialyses each performed in a dialysis tube with acutoff threshold being 3 kDa, at 4° C., against 200 ml of phosphatebuffer, pH 7.6 10 mM.

Example 9 Preparation of Murine KLH-TNFa Heterocomplex

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccinee the production of antibodies neutralizing themurine TNFα.

0.625 mg of KLH protein is dissolved in 0.6 ml of 10 mM borate buffer,pH 8.8, 150 mM NaCl. To this solution is added 1 mg of human IFNαprotein dissolved in 1 ml of the same buffer.

The thus obtained protein mixture is treated using glutaraldehyde in thefinal concentration of 0.026 M for 45 minutes at room temperature.

The excess glutaraldehyde is then removed by 3 successive 4 hourdialyses each performed in a dialysis tube with a cutoff threshold being3 kDa, at 4° C., against 200 ml of phosphate buffer, pH 7.6 10 mM 150 mMNaCl.

The mixture is then treated using formaldehyde at the finalconcentration of 33 mM for 48 hours. Then the reaction is blocked by theaddition of final 0.1M glycine for 1 hour at room temperature. Themixture is finally dialyzed under the same conditions as the previouslyperformed dialysis.

Example 10 Preparation of a Tat Peptide Heterocomplex (19-50)-_(h)IgE

The Tat peptide (19-50) brings auxiliary helper epitopes.

Sequence of the Tat peptide to be used:

Lys-Thr-Ala-Cys-Thr-Asn-Cys-Tyr-Cys-Lys-Lys-Cys-Cys-Phe-His-Cys-Gln-Val-Cys-Phe-lle-Thr-Lys-Ala-Leu-Gly-lle-Ser-Tyr-Gly-Arg-Lys

Such a conjugate is the active ingredient of a vaccine able to mainlyinduce in the vaccinee the formation of (human) anti-IgE antibodies.

0.1 mg of Tat peptide (19-50) (4.06×10⁻⁸ mole) are dissolved in 0.2 mlof 10 mM borate buffer, pH 8.5. To this solution is added 1 mg of humanIgE (6.6×10⁻⁹ mole) dissolved in 1 ml of the same buffer.

The thus prepared mixture is then treated using formaldehyde at thefinal concentration of 0.026 M for 30 minutes at room temperature.

The excess glutaraldehyde is then removed by 2 successive 4 hourdialyses and a final 16 hour dialysis against a large volume ofphosphate buffer, 10 mM, pH 7.4 containing 0.8% of NaCl (PBS), at 4° C.,using a dialysis bag with a cutoff threshold being at 10 kDa. (IgEpeptide ratio=50:1)

Exemple 11 Preparation of a Heterocomplex Against the Ricin Fragment

Such a heterocomplex is the active ingredient of a vaccine able toinduce in the vaccinee the formation of neutralizing antibodies directedagainst the fragment involved in the binding of the ricin molecule,thereby preventing it from exerting its toxic activity.

To 0.5 mg of KLH (1.1×10⁻³ mole) dissolved in 0.5 ml of 10 mM phosphatebuffer, pH 8.2, is added 1 mg (3.3×10⁻⁸ mole) of a fragment of ricindissolved in 1 ml of the same buffer. The thus prepared mixture istreated using formaldehyde at the final concentration of 0.026 M for 30minutes at room temperature.

The excess glutaraldehyde is then removed by 2 successive 4 hour eachdialyses and a 16 hour dialysis against a large volume of phosphatebuffer, 10 mM, containing 0.8% of NaCl (PBS), using a dialysis bag witha cutoff threshold being 3 kDa. (KLH:Ricin-a ratio=1:30).

Examples of Biochemical Characterizations of Heterocomplexes

A. Material and Methods of Examples 12 to 23

The biochemical characterizations of heterocomplexes are performed bymeans of the following techniques:

1. Antigenicity Test

The study of antigenicity of a heterocomplex compared to theantigenicity of poteins making it up is performed by a conventionalindirect ELISA. Such a technique allows for a protein to bequantitatively measured through the specific recognition of an antibodyraised against an antigen. Such a test comprises depositing sampledilutions containing the sought protein in wells of a microtiter plate.A specific polyclonal antibody reacts with the immobilized protein. Asecond antibody, conjugated with horseradish peroxydase, specific to thefirst one is then added. The formed complex is revealed throughincubation with OPD. The resulting yellow colour is directlyproportional to the amount of bound proteins. The absorbance (DO) ofeach well is measured by means of a microplate reader. The amount ofprotein present in the sample is then determined by means of acalibrating range.

2. Isoelectrofocusing in Agarose Gel Followed by a Western Blot

The isoelectrofocusing in agarose gel allows to separate moleculesdepending on their isoelectric point (Ip) under non denaturingconditions, allowing to study heterocomplexes without destroying theweak bonds existing within such complexes.

The isofocusing is followed by an emergence through a Western blot.After electrophoretic separation, the molecules are transferred on anitrocellulose membrane through capillarity. Such molecules are thencharacterized by immunochemistry.

3. Measurement of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule (First Method)

Estimating the percentage of molecules of the antigen of interestcovalently linked to carrier protein molecules in an immunogenic productoccurs, for example, through molecular sieving on a column containingSuperdex 200, under denaturing (8 M urea) and reducing (5%beta-mercaptoethanol) conditions.

The % of covalently linked molecules of the antigen of interest isdeducted from the amount of antigen of interest (determined by aconventional indirect ELISA) present in the exclusion volume of thecolumn. Indeed, the carrier protein molecule, such as KLH, having amolecular mass much higher than the highest fractionating limit of thecolumn being used (200 kDa in this case), exists in the exclusion volumeof such a column. Thus, under denaturing and reducing conditions, onlythe antigens of interest covalently linked to the carrier proteinmolecule exist in the exclusion volume.

4. Measurement of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule (Second Method)

The percentage of cytokin fixed on the carrier protein (KLH) wasdetermined by a double sandwich ELISA, by means of a capture antibodyspecifically directed against the carrier protein.

100 μl of horse polyclonal antibodies raised against KLH (1 mg/ml)diluted in a 10 mM phosphate buffer, pH 7.3 NaCl 150 mM (PBS) are boundin wells of a microtiter plate (high-binding Costar) for 2 hours at 37°C. After 3 washes in PBS/0.1% Tween 20 (PBST), the wells are saturatedwith PBS containing 2% of BCS.

After 1.30 hour of saturation, the wells are washed three times withPBST, then heterocomplex 2 by 2 dilutions (10, 5, 2.5, 1.25, 0.625,0.312 and 0.156 μg/ml) made in duplicate, are added in the wells (100μl/well).

After 2 hours of incubation, the wells are washed three times with PBST.The Tween, a dissociating agent, present in the washing buffer, allowsto remove all the molecules which are not covalently linked to the KLHbeing, itself, specifically bound on the capture antibody.

Then, both heterocomplex dilutions are treated in two different ways:

a) the first set is incubated with an antibody raised against KLH

b) the second set is incubated with an antibody raised against cytokin.

After 1.30 hour of incubation at 37° C., the wells are washed aspreviously indicated then incubated with a secondary antibody coupled tothe peroxydase, directed againt the origin species of the firstantibody. After 1.30 hour of incubation at 37° C., the antibodies arewashed again. Then, the addition of the peroxydase substrate,O-PhenyleneDiamine (OPD) allows for the revealation of the presence ofthe KLH bound by the capture antibody and cytokins covalently bound onthe KLH.

The amount of KLH bound by the capture antibody and then the amount ofcytokin molecules covalently bound on the KLH are calculated by means ofcalibrating curves done by ELISA.

The percentage of cytokin covalently bound to the KLH is thendetermined.

Example 12 Biochemical Characterization of the KLH-Murine VEGFHeterocomplex

1. Antigenicity

The KLH-murine VEGF heterocomplex has an antigenicity identical to theantigenicity of murine VEGF.

2. Isoelectrofocusing in Agarose Gel Followed by a Western Blot

FIG. 1 shows that the KLH-murine VEGF heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it up.

Example 13 Biochemical Characterization of the KLH-Human VEGFHeterocomplex

1. Antigenicity

The KLH-human VEGF heterocomplex has an antigenicity identical to theantigenicity of human VEGF.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 2 shows that the KLH-human VEGF heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it up. The human VEGF sample was deposited at three differentlocations in order to show that it still migrates at the same location.

Example 14 Biochemical Characterization of KLH-Murine IL4 Heterocomplex

1. Antigenicity

The KLH-murine IL4 heterocomplex has an antigenicity identical to theantigenicity of murine IL4.

Example 15 Biochemical Characterization of the KLH-Human IL4Heterocomplex

1. Antigenicity

The human KLH-IL4 complex has an antigenicity equal to the antigenicityof the human IL4 protein.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 3 shows that the human KLH-IL4 heterocomplex migrates under theform of a single strip with a Ip different from the native moleculesmaking it up.

Example 16 Biochemical Characterization of the KLH-IFNα Heterocomplex

1. Antigenicity

The human KLH-IFNα complex has an antigenicity identical to theantigenicity of the human IFNα.

2. Estimation of the Percentage of Molecules of the Antigen of InterestCovalently Linked to the Carrier Protein Molecule

The KLH-IFNα preparation is passed on a superdex S200 column followingthe above described conditions. The apparent peak in the exclusionvolume was collected, dialyzed then freeze-dried. The concentration inantigen of interest was determined by the indirect ELISA technique. TheIFNα amount present in the excluded volume is 30 μg while 1000 μg of IFNwere used for preparing the immunogenic product, without any measurableloss of antigen during the preparing method. The percentage of moleculesof the antigen of interest covalently linked to the KLH molecule in theimmunogenic product comprising KLH-IFNα heterocomplexes can therefore beestimated to approximately 3%.

Example 17 Biochemical Characterization of the gp160-IFNα Heterocomplex

1. Antigenicity

The human gp160-IFNα complex has an antigenicity dentical to theantigenicity of the gp160 protein as well as to that of human IFNα.

2. Isoelectrofocusing Followed by a Western Blot

FIG. 4 shows that the human gp160-IFNα heterocomplex migrates under theform of one single strip at a Ip being quite different from the Ip ofthe gp160 protein recombining the component. The Ip of such aheterocomplex is slightly lower than that of IFNα.

Example 18 Biochemical Characterization of the gp160-Toxoid TatHeterocomplex

1. Antigenicity

The gp160-toxoid Tat complex has an antigenicity identical to theantigenicity of the gp160 protein and to that of the Tat protein.

Example 19 Biochemical Characterization of the gp160-GM TatHeterocomplex

1. Antigenicity

The gp160-GM Tat complex has an antigenicity identical to theantigenicity of the gp160 protein and to that of the Tat protein.

Example 20 Biochemical Characterization of the KLH-Murine IL4Heterocomplex

1. Antigenicity

The murine KLH-IL4 heterocomplex has an antigenicity identical to theantigenicity of murine IL4.

2. Estimation of the % of Antigen Molecules of Interest Covalently Boundto the Carrier Protein Molecule

11% of molecules of murine IL4 are covalently fixed to the KLH.

Example 21 Biochemical Characterization of the KLH-IFNα Heterocomplex

1. Antigenicity

The KLH-human IFNα complex has an antigenicity identical to theantigenicity of the human IFNα.

2. Estimation of the % of Antigen Molecules of Interest Covalently Fixedto the Carrier Protein Molecule

8% of molecules of human IFNα are covalently bound to the KLH.

Example 22 Biochemical Characterization of the Tat-_(h)IgE PeptideHeterocomplex

1. Antigenicity

The Tat-_(h)IgE peptide complex has an antigenicity comparable to thatof human IgE.

Example 23 Biochemical Characterization of the KLH-β Ricin-Heterocomplex

1. Antigenicity

The KLH-β Ricin complex has an antigenicity comparable to that of the βricin fragment.

2. Isoelectrofocusing Followed by a Western Blot

The complex migrates under the form of a single strip and the presenceof the β fragment is enhanced by Western Blot.

Examples of Immunogenic Activity of Heterocomplexes

Example 24 Immunogenic Activity of the KLH-Murine VEGF Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-murine VEGF preparation compared tothat of the murine VEGF was studied in 18 to 20 g balb c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without any immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is studied in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bycell proliferation test conducted on PBMCs cultivated in the presence ofthe complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice immunized both with the KLH-murine VEGF preparation and themurine VEGF only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine VEGF do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the murine VEGF, determinedby ELISA and expressed in titer (opposite of the dilution giving anoptical density higher than 0.3). FIG. 5 shows the resulting antibodytiters.

The mice immunized with the KLH-murine VEGF preparation show higherantibody titers of the IgG type than those of mice immunized with themurine VEGF only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of VEGF, selective growth factor ofendothelial cells. Endothelial cells (HUVECs) are cultivated in flatbottom wells of a microculture plate at a level of 3,000 cells per well.The sera of each group of mice were pooled. Different dilutions of suchserum pools (1/100-1/800) taken at D-2 and D72 were pre-incubated for 2hours with 20 ng/ml of murine VEGF then deposited on such endothelialcells. The cell culture continued at 37° C. in a humid atmosphere loadedwith 5% of CO2 for 3 days. 18 hours before the end of the incubation,0.5 μCi of titered thymidine/well were added. The neutralizing seraprevent the murine VEGF from inducing the proliferation of endothelialcells, while non neutralizing sera allow for the proliferation of suchcells. The results are expressed in neutralization percentage. FIG. 6shows the obtained results.

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the murine VEGF.

Example 25 Immunogenic Activity of the KLH-Human VEGF Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-human VEGF preparation compared tothat of the human VEGF was studied in 18 to 20 g balb c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice immunized both with the murine KLH-VEGF preparation and themurine VEGF only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human VEGF do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the human VEGF, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). FIG. 7 shows the resulting antibody titers.

The mice immunized with the KLH-human VEGF preparation show higherantibody titers of the IgG type than those of mice immunized with thehuman VEGF only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of VEGF, selective growth factor ofendothelial cells. Endothelial cells (HUVECs) are cultivated in flatbottom wells of a microculture plate at a level of 3,000 cells per well.The sera of each group of mice were pooled. Different dilutions of suchserum pools (1/100-1/800) taken at D-2 and D72 were pre-incubated for 2hours with 20 ng/ml of human VEGF then deposited on such endothelialcells. The cell culture is continued at 37° C. in a humid atmosphereloaded with 5% of CO2 for 3 days. 18 hours before the end of theincubation, 0.5 μCi of titered thymidine/well were added. Theneutralizing sera prevent the human VEGF from inducing the proliferationof endothelial cells, while non neutralizing sera allow for theproliferation of such cells. The results are expressed in neutralizationpercentage. FIG. 8 shows the obtained results.

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human VEGF.

Example 26 Immunogenic Activity of the KLH-Murine IL4 Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the murine KLH-IL4 preparationcompared to that of the murine IL4 was studied in 18 to 20 g balb cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 8 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2 and D72.

3 control mice receive the same preparations without an immunogen.

14 days after the last immunization, the control mice and the miceimmunized with the KLH-murine IL4 were challenged with birch-tree pollenin the presence of alum (100 μg/mice) through the subcutaneous route atD74, D95 and D109. Blood samples are regularly taken in order to followthe occurence of classe G and E antibodies raised against Bet v 1, amajor allergen of the birch-tree pollen.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the KLH-murine IL4 preparation and themurine IL4 only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine IL4 do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the murine VEGF, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). FIG. 9 shows the resulting antibody titers.

The mice immunized with the KLH-murine IL4 preparation show higherantibody titers of the IgG type than those of mice immunized with themurine IL4 only.

The neutralizing activity of those antibodies present in mice immunizedwith the KLH-murine IL4 preparation was measured by means of thebiological activity test of the murine IL4. This test uses HT-2 cells,murine cell lineages the growth of which is IL4 murine-dependent(Watson, J. 1979. J. Exp. Med. 150:1510.). Endothelial cells HT-2 arecultivated in round bottom wells of a microculture plate at a level of10,000 cells per well. Sera diluted at 1/50 taken at D-2 and D72 arepre-incubated for 2 hours with 50 ng/ml of murine H4 then deposited onHT-2 cells. The cell culture is continued at 37° C. in a humidatmosphere loaded with 5% of CO2 for 3 days. 4 hours before the end ofthe incubation, 0.5 μCi of titered thymidine/well were added. Theneutralizing sera prevent the murine IL4 from inducing the proliferationof HT-2 cells, while non neutralizing sera allow for the proliferationof such cells. The results are expressed in neutralization percentage.FIG. 10 shows the obtained results.

The antibodies induced by the complex are neutralizing.

Moreover, such neutralizing antibodies raised against murine IL4 preventthe production, by those mice, of antibodies of the IgE type raisedagainst Bet v 1, when the latter are challenged with birch-tree pollen.FIG. 11 indeed shows that mice immunized with KLH-murine IL4 haveneutralizing IgGs raised against murine NL4 blocking the production ofIgE raised against Bet v 1 and start to produce antibodies of the IgGtype directed against Bet v 1. On the other hand, mice which did notreceive any murine KLH-IL4 and therefore not having any antibodies ofthe IgG type directed against IL4, only produce antibodies of the IgEtype directed against Bet v 1.

Example 27 Immunogenic Activity of the KLH-Human IL4 Heterocomplex

A. Material and Methods

The immunogenic (humoral) of the KLH-human IL4 preparation compared tothat of the human IL4 was studied in 18 to 20 g balb c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1. Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the KLH-human IL4 preparation and the humanIL4 only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IL4 do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IL4, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). TABLE 1 Titer D-2 D72 Control mice: Controlmouse 1 <500⁻¹ <500⁻¹ Control mouse 2 <500⁻¹ <500⁻¹ Control mouse 3<500⁻¹ <500⁻¹ Mice immunized with human IL4: mouse 4 <500⁻¹ 32,000⁻¹mouse 5 <500⁻¹ 48,000⁻¹ mouse 6 <500⁻¹ 16,000⁻¹ Mice immunized with theKLH-human IL4 complex: mouse 7 <500⁻¹ 256,000⁻¹ mouse 8 <500⁻¹ 128,000⁻¹mouse 9 <500⁻¹ 128,000⁻¹

The mice immunized with the KLH-human IL4 preparation show higherantibody titers of the IgG type than those of mice immunized with thehuman IL4 only.

The neutralizing activity of such antibodies induced by the humanKLH-IL4 preparation was measured by means of the biological activitytest of human IL4. This test uses TF-1 cells, human cell lineage thegrowth of which is IL4 human-dependent (Kitamura, T. et al., 1989. J.Cell Physiol. 140:323-34). TF-1 cells are cultivated in round bottomwells of a microculture plate at a level of 10,000 cells per well. Seradiluted at 1/50 taken at D-2 and D72 preincubated for 2 hours with 50ng/ml of humain IL4 were then deposited on the TF-1 cells. The cellculture is continued at 37° C. in a humid atmosphere loaded with 5% ofCO2 for 3 days. 4 hours before the end of the incubation, 0.5 μCi oftitered thymidine/well were added. The neutralizing sera prevent thehuman IL4 from inducing the proliferation of TF-1 cells, while nonneutralizing sera allow for proliferation of such cells. The results areexpressed in neutralization percentage. FIG. 12 shows the obtainedresults.

The antibodies induced by the complex are neutralizing.

Example 28 Immunogenic Activity of the KLH-IFNα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-human IFNα preparationcompared to that of the human IFNα was studied in 18 to 20 g balb cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the KLH-human IFNα preparation and thehuman IFNα only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders. systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human NFNα, determinedby ELISA and expressed in titer (opposite of the dilution giving anoptical density higher than 0.3). The table 2 shows the resultingantibody titers. TABLE 2 Titer D-2 D72 Control mice: Control mouse 1<500⁻¹ <500⁻¹ Control mouse 2 <500⁻¹ <500⁻¹ Control mouse 3 <500⁻¹<500⁻¹ Mice immunized with IFNα: mouse 4 <500⁻¹ 96,000⁻¹ mouse 5 <500⁻¹128,000⁻¹ mouse 6 <500⁻¹ 96,000⁻¹ Mice immunized with the KHL-IFNαcomplex: mouse 7 <500⁻¹ 96,000⁻¹ mouse 8 <500⁻¹ 96,000⁻¹ mouse 9 <500⁻¹128,000⁻¹

The mice immunized with the KHL-human IFNα preparation show antibodytiters of the IgG type equivalent to those of mice immunized with thehuman IFNα only.

The neutralizing activity of such antibodies was measured by means ofthe biological activity test of the human IFNα. (Rubinstein S, J Viral,1981, 755-8). The aim of this test for measuring the antiviral effect isto evaluate the inhibition of the MDBK cell lysis by the VSV (VesicularStomatitis virus) in the presence of IFN. MDBK cells are cultivated inround bottom wells of a microculture plate at a level of 350,000 cellsper well. Different dilutions of sera (1/100 at 1/800) taken at D-2 andD72 were pre-incubated for 2 hours with 5 ng/ml of human IFNα thendeposited on MDBK cells. After 20 hours of cell culture performed at 37°C. in a humid atmosphere loaded with 5% of CO2, the diluted sera presentin the wells are removed, the cells washed, then 100 μl containing 100LD50 (50% lethal dosis) of VSV virus are added. 18 hours after theaddition of the virus the lytic effect of the virus is measured. Theneutralizing sera allow the VSV to lyse cells, while non neutralizingsera prevent such a lysis. The results are expressed in neutralizationpercentage. TABLE 3 1/100 1/200 1/400 1/800 Mice immunized with IFNα:mouse 4 D-2 0 0 0 0 D72 100 75 65 50 mouse 5 D-2 0 0 0 0 D72 100 67 6055 mouse 6 D-2 0 0 0 0 D72 100 72 65 60 Mice immunized with the KLH-IFNαconjugate mouse 7 D-2 0 0 0 0 D72 100 100 100 100 mouse 8 D-2 0 0 0 0D72 100 100 100 100 mouse 9 D-2 0 0 0 0 D72 100 100 100 100

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human IFNα. The results are expressed inneutralization percentage.

Example 29 Immunogenic Activity of the gp160-IFNα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the human gp160-IFNα preparationcompared to that of the human IFNα was studied in 18 to 20 g balb cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

1—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice immunized both with the gp 160-human IFNα preparation and thehuman IFNα only, do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-human IFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IFN, determined byELISA and expressed in titer (opposite of the dilution giving an opticaldensity higher than 0.3). TABLE 4 Titer D-2 D72 Control mice: Controlmouse 1 <500⁻¹ <500⁻¹ Control mouse 2 <500⁻¹ <500⁻¹ Control mouse 3<500⁻¹ <500⁻¹ Mice immunized with IFNα: mouse 4 <500⁻¹ 64,000⁻¹ mouse 5<500⁻¹ 96,000⁻¹ mouse 6 <500⁻¹ 128,000⁻¹ Mice immunized with thegp160-IFNα complex: mouse 7 <500⁻¹ 96,000⁻¹ mouse 8 <500⁻¹ 96,000⁻¹mouse 9 <500⁻¹ 64,000⁻¹

The mice immunized with the gp 160-human IFNα preparation present IgGtype antibody titers equivalent to those of mice immunized with thehuman IFNα only.

The neutralizing activity of such antibodies has been measured with thehelp of the human IFNα bilogical activity test described in the formerexample. Results are given in neutralization %. TABLE 5 1/100 1/2001/400 1/800 Mice immunized with the IFNα: mouse 4 D-2 0 0 0 0 D72 100 8070 53 mouse 5 D-2 0 0 0 0 D72 100 70 65 50 mouse 6 D-2 0 0 0 0 D72 10065 60 57 Mice immunized with the gp160-IFNα conjugate: mouse 7 D-2 0 0 00 D72 100 100 100 100 mouse 8 D-2 0 0 0 0 D72 100 100 100 100 mouse 9D-2 0 0 0 0 D72 100 100 100 100

The antibodies induced by the complex have a higher neutralizing powerthan that induced by the human IFNα.

Example 30 Immunogenic Activity of the gp160-Toxoid Tat Heterocomplex

A. Material and Methods

The immunogenic (humoral and cellular) activity of the gp160-toxoid Tatpreparation compared to that of the toxoid Tat was studied in 18 to 20 gbalb c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D-2.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results:

1—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice immunized both with the gp160-toxoid Tat preparation and thetoxoid Tat only do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-toxoid Tat do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the Tat, determined by ELISAand expressed in titer (reciprocal of the dilution giving an opticaldensity higher than 0.3). Table 6 shows the resulting antibody titers.TABLE 6 Titer Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹ <500⁻¹Control mouse 2 <500⁻¹ <500⁻¹ Control mouse 3 <500⁻¹ <500⁻¹ Miceimmunized with toxoid Tat: mouse 4 <500⁻¹ 48,000⁻¹ mouse 5 <500⁻¹64,000⁻¹ mouse 6 <500⁻¹ 48,000⁻¹ Mice immunized with gp160-toxoid Tatconjugate: mouse 7 <500⁻¹ 64,000⁻¹ mouse 8 <500⁻¹ 128,000⁻¹ mouse 9<500⁻¹ 64,000⁻¹

The mice immunized with the gp160-toxoid Tat preparation show higherantibody titers of the anti-Tat IgG type than those of mice immunizedwith the toxoid Tat only.

The neutralizing activity of such antibodies was measured by means ofthe Cat assay. Different dilutions of sera (1/100-1/800) taken at D-2and D72 are incubated for 2 hours with 50 ng/ml of native Tat. Suchdilutions are then deposited on HeLa cells, stably infected cells with aplasmid containing LTR of the VIH-1 as the promotor of theChloramphenicol Acetyl transferase gene (CAT). After 24 hours ofculture, the cells are lyzed and the amount of CAT protein produced ismeasured by an ELISA test, the Cat assay (Boehringer Mannheim).Neutralizing sera prevent the Tat protein from inducing the expressionof the CAT protein, while the non neutralizing sera allow for thesynthesis of such CAT protein. The results are expressed inneutralization %. TABLE 7 1/100 1/200 1/400 1/800 Mice immunized withtoxoid Tat: mouse 4 D-2 0 0 0 0 D72 60 50 25 20 mouse 5 D-2 0 0 0 0 D7260 55 30 20 mouse 6 D-2 0 0 0 0 D72 65 50 30 30 Mice immunized withgp16-toxoid Tat conjugate: mouse 7 D-2 0 0 0 0 D72 100 100 100 100 mouse8 D-2 0 0 0 0 D72 100 100 100 100 mouse 9 D-2 0 0 0 0 D72 100 100 100100

The antibodies induced by the gp160-toxoid Tax conjugate have a higherneutralizing power than that induced by the toxoid Tat

2. Production of MIP1α

The production of MIP1α in culture supernatants of splenocytes.Splenocytes of immunized mice and control mice are isolated thencultivated in round bottom wells of a micro-culture plate at a level of100,000 cells/well in the presence of 5 Mg/ml of p24, gp160, native Tatand a mixture of 5 μg/ml gp160 and 5 μg/ml of native Tat. Thesupernatants are taken after 24 hours of culture and the presence ofMIP1 in the supernatants is measured by means of a R&D ELISA test. Theresults are expressed in μg/ml. TABLE 8 Gp160 + native native Gp160 TatTat P24 Control mice: mouse 1 MIP1α 95 90 145 9 D72 mouse 2 Mip1α 100 90136 7 D72 mouse 3 MIP1α 120 110 132 9 D72 Mice immunized with toxoidTat: mouse 4 MIP1α 145 130 190 7 D72 mouse 5 MIP1α 128 145 225 9 D72mouse 6 MIP1α 150 230 295 10 D72 Mice immunized with gp160-toxoid Tatconjugate: mouse 7 MIP1α 875 736 1725 9 D72 mouse 8 MIP1αα 945 905 19007 D72 mouse 9 MIP1α 1025 795 1755 8 D72

Splenocytes of mice immunized with the gp160-toxoid Tat conjugateproduce more MIP1α chemiokins than cells of mice immunized by the toxoidTat only when they are activated, in vitro, by the immunogens usedduring the immunization.

4. Proliferation of Splenocytes of Immunized Mice (CMI Test)

Splenocytes of immunized mice and of control mice are isolated thencultivated in round bottom wells of a micro-culture plate at a level of100,000 cells/well in the presence of p24, gp160, native Tat and amixture of gp160 and native Tat. The cell culture is continued at 37° C.in a humid atmosphere loaded with 5% of CO2 for 6 days. 18 hours beforethe end of the incubation, 0.5 μCi of titered thymidine/well were added.The intensity of the immune response is proportional to theproliferation index Ip.Ip=spm (strokes per minute) for the given antigen/control spm TABLE 9native Gp160+ Gp160 Tat native Tat P24 Control mice: mouse 1 1.1 1.1 11.2 D72 mouse 2 1 1.1 1.1 1.1 D72 mouse 3 1.2 1 1 1.1 D72 Mice immunizedwith toxoid Tat: mouse 4 1.2 8 10 1.1 D72 mouse 5 1 9 9 1.2 D72 mouse 61 10 9 1.2 D72 Mice immunized with gp160-toxoid Tat conjugate: mouse 7 911 8 1 D72 mouse 8 10 9 7.5 1 D72 mouse 9 10.5 9 8 1 D72

Splenocytes of mice immunized with the gp160-toxoid Tat conjugate or thetoxoid Tat, proliferate, when they are activated, in vitro, with theimmunogens used during the immunization.

Example 31 Immunogenic Activity of the gp160-GM Tat Heterocomplex

A. Material and Methods

The immunogenic (humoral and cellular) activity of the gp160-GM Tatpreparation compared to that of the toxoid Tat was studied in 18 to 20 gbalb c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an emulsion in AIF through the intramuscular route. A 5 μgbooster injection in AIF is given at D-2.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (100μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice immunized both with the gp160-GM Tat preparation and the toxoidTat only, do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofgp160-toxoid Tat do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 100 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the Tat, determined by ELISAand expressed in titer (reciprocal of the dilution giving an opticaldensity higher than 0.3). Table 10 shows the resulting antibody titers.TABLE 10 Titer D-2 D72 Control mice: Control mouse 1 <500⁻¹ <500⁻¹Control mouse 2 <500⁻¹ <500⁻¹ Control mouse 3 <500⁻¹ <500⁻¹ Miceimmunized with GM Tat: mouse 4 <500⁻¹ 64,000⁻¹ mouse 5 <500⁻¹ 64,000⁻¹mouse 6 <500⁻¹ 48,000⁻¹ Mice immunized with the gp160-GM Tat conjugate:mouse 7 <500⁻¹ 128,000⁻¹ mouse 8 <500⁻¹ 128,000⁻¹ mouse 9 <500⁻¹64,000⁻¹

The mice immunized with the gp160-GM Tat preparation show higherantibody titers of the anti-Tat IgG type than those of mice immunizedwith the GM Tat only.

Example 32 Immunogenic Activity of the KLH-Murine TNFα Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-murine TNFα preparationcompared to that of the murine TNFα was studied in 18 to 20 g balb/cmouse.

At day 0, a group of 3 mice (group A) receives a 0.1 ml injection of anAIF emulsion through the intramuscular route containing 60 μg of theKLH-TNFα complex. A booster injection of 30 μg and 15 μg in AIF is givenrespectively at D21 and D60. 3 control mice receive a dosis equivalentin murine TNFα according to the same protocol. (group B)

At day 0, a group of 3 mice (group C) receives a 0.1 ml injection in AIFthrough intramuscular route containing 60 μg of KLH-murine TNFαheterocomplex and 30 μg of the phosphorothioate oligodeoxynucleotide5′-TCCATGACGTTCCTGACGTT-3′ (CpG ADN: 1826). A booster injection of 30 μgand of 15 μg of the KKL-murine TNFα heterocomplex in AIF is givenrespectively at D21 and D60. 3 control mice receive a dosis equivalentin murine TNFα according to the same protocol. (group D)

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at d-2.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the murine KLH-TNFα preparation and themurine TNFα only do not show any clinical sign and no anatomic wound.The immunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-murine TFNα do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex with orwithout the DNA CPG 1826 show any sign of toxicity (temperature,cutaneous disorders, systemic or regional signs) during the 7 daysfollowing the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type raised against the murine TNFα, determined byELISA and expressed in titer. The presence of antibodies of the IgA typedirected against the murine TNFα in vaginal secretions was alsodetermined by ELISA and expressed in titer. The titer represents theopposite of the dilution giving an optical density higher than 0.3. Thefollowing table shows the resulting antibody titers. TABLE 11 VaginalIgA D-2 D72 D-2 D72 Mice immunized with KLH-murine TNFα (group A) 1<500⁻¹ 64,000⁻¹ <10⁻¹ 20⁻¹ 2 48,000⁻¹ 20⁻¹ 3 64,000⁻¹ 40⁻¹ Miceimmunized with the Murine TNFα (group B) 4 <500⁻¹ 750⁻¹ <10⁻¹ 10⁻¹ 51,000⁻¹ 20⁻¹ 6 750⁻¹ 10⁻¹ Mice immunized with KLH-murine TNFα in thepresence of CPG (group C) 7 <500⁻¹ 128,000⁻¹ <10⁻¹ 160⁻¹ 8 256,000⁻¹80⁻¹ 9 256,000⁻¹ 320⁻¹ Mice immunized with murine TNFα: in the presenceof CpG (group D) 7 <500⁻¹ 2000⁻¹ <10⁻¹ 20⁻¹ 8 4,000⁻¹ 40⁻¹ 9 3,000⁻¹40⁻¹

Example 33 Immunogenic Activity of the Tat Peptide-_(h)IgE Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-human IgE preparationcompared to that of the human IgE was studied in 18 to 20 g balb cmouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dosis (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

2—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the KLH-human IgE preparation and the humanIgE only, do not show any clinical sign and no anatomic wound. Theimmunosuppression test shows that doses of 100 ng/ml to 1 μg/ml ofKLH-human IgE do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the human IgE, determined byELISA and expressed in titer (reciprocal of the dilution giving anoptical density higher than 0.3). The following table shows theresulting antibody titers. TABLE 12 Titer D-2 D72 Control mice: 1 <500−¹<500−¹ 2 3 Mice immunized with _(h)IgE 4 <500⁻¹ 64,000⁻¹ 5 128,000⁻¹ 6128,000⁻¹ Mice immunized with KLH-_(h)IgE: 7 <500⁻¹ 256,000⁻¹ 8128,000⁻¹ 9 256,000⁻¹

The mice immunized with the KHL-_(h)IgF preparation show antibody titersof the IgG type slightly higher than those of mice immunized with thehlgE preparation only.

Example 34 Immunogenic Activity of the KLH-Ricin-β Heterocomplex

A. Material and Methods

The immunogenic (humoral) activity of the KLH-Ricin-β preparationcompared to that of the ricin β fragment was studied in 18 to 20 gbalb/c mouse.

1—Immunization

At days 0, 7, 14, 21, a group of 3 mice receives a 0.1 ml (10 μg)injection of an AIF emulsion through the intramuscular route. A 5 μgbooster injection in AIF is given at D60.

A blood sample at the retro-orbital level is taken from each mousebefore the first injection at D-2.

3 control mice receive the same preparations without an immunogen.

The mice are sacrificed 12 days after the last immunization.

2—Toxicity

The abnormal toxicity is sought in 3 mice receiving one human dose (50μg) according to the pharmacopeia.

The lack of immunotoxicity of the heterocomplex is evaluated in vitro bya cell proliferation test conducted on PBMCs cultivated in the presenceof the complex and stimulated by PPD or toxoid tetanos.

B. Results

3—Lack of Toxicity of the Heterocomplex In Vivo and In Vitro

The mice both immunized with the human KLH-Ricin-preparation and thericin β-fragment only do not show any clinical sign and no anatomicwound. The immunosuppression test shows that doses of 100 ng/ml to 1μg/ml of KLH-ricin β do not reduce the proliferation of lymphocytes.

None of the three mice immunized with 50 μg of the heterocomplex showany sign of toxicity (temperature, cutaneous disorders, systemic orregional signs) during the 7 days following the injection.

2—Humoral Response

The humoral response is measured by the presence in the serum ofantibodies of the IgG type directed against the β fragment of ricin,determined by ELISA and expressed in titer (reciprocal of the dilutiongiving an optical density higher than 0.3). The following table showsthe resulting antibody titers. TABLE 13 Titer D-2 D72 Control mice: 1<500⁻¹ <500⁻¹ 2 3 Mice immunized with ricin-β 4 <500⁻¹ 256,000⁻¹ 5512,000⁻¹ 6 256,000⁻¹ Mice immunized with KLH-Ricin-β 7 <500⁻¹ 256,000⁻¹8 256,000⁻¹ 9 128,000⁻¹

The neutralizing activity of such antibodies was checked by theinjection to the mouse of mixtures of anti-Ricin-β and ricin serum whichdid not cause the animal's death, contrary to what was observed duringthe administration to the mouse of ricin and normal serum mixtures.

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1. A stable immunogenic product for inducing antibodies raised againstone or more antigenic proteins in a subject, characterized in that itcomprises protein immunogenic heterocomplexes consisting of associationsbetween (i) antigenic protein molecules and (ii) carrier proteinmolecules and in that less than 40% of the antigenic proteins (i) arecovalently linked to carrier protein molecules (ii).
 2. An immunogenicproduct according to claim 1, characterized in that each heterocomplexcomprise (i) a plurality of antigenic proteins linked to (ii) a carrierprotein molecule.
 3. An immunogenic product according to claim 2,characterized in that, for each immunogenic heterocomplex, the pluralityof antigenic proteins (i) is made up of a plurality of specimens of asingle antigenic protein.
 4. An immunogenic product according to claim2, characterized in that, for each immunogenic heterocomplex, theantigenic proteins (i) consist of a plurality of specimens of a proteinbeing normally recognized as a self protein by the cells of saidsubject's immune system.
 5. A product according to claim 1,characterized in that it comprises 5 to 50 antigenic proteins (i) forone carrier protein molecule (ii), preferably 20 to 40 antigenicproteins (i) for one carrier protein molecule (ii).
 6. An immunogenicproduct according to claim 1, characterized in that the covalent bondsbetween one or more antigenic proteins (i) and the carrier proteinmolecule (ii) are made through a bifunctional bond chemical agent.
 7. Animmunogenic product according to claim 6, characterized in that saidbinding chemical agent comprises at least two free aldehyde functions.8. An immunogenic product according to claim 7, characterized in thatsaid binding chemical agent is glutaraldehyde.
 9. An immunogenic productaccording to claim 1, characterized in that the antigenic protein(s) (i)consist(s) in cytokins naturally produced by said subject.
 10. Animmunogenic product according to claim 9, characterized in that theantigenic protein(s) (i) is/are selected amongst interleukin-4, alphainterferon, gamma interferon, VEGF, interleukin-10, TNF alpha, TGF beta,interleukin-5 and interleukin-6.
 11. An immunogenic product according toclaim 1, characterized in that the antigenic protein(s) (i) is/areselected amongst the papillomavirus E7 protein, the HIV 1 virus Tatprotein, the HTLV 1 or HTLV 2 virus Tax protein and the self p53protein.
 12. An immunogenic product according to claim 1, characterizedin that the antigenic protein(s) is/are selected amongst proteins lethalto man at a dosis lower than 1 mg.
 13. An immunogenic product accordingto claim 12, characterized in that the antigenic protein(s) (i) isselected amongst ricin, botulic toxins, staphylococcus enterotoxins aswell as an anthrax toxic protein.
 14. An immunogenic product accordingto claim 1, characterized in that the carrier protein molecule (ii) isan immunogenic protein inducing the production of cytotoxic lymphocytesraised against cells having at their surface said carrier proteinmolecule or any peptide being derived from it, in association with ClassI molecules of the Major Histocompatibility Complex (MHC).
 15. Animmunogenic product according to claim 14, characterized in that thecarrier protein molecule (ii) is selected amongst papillomavirus L1, L2and E7 proteins.
 16. An immunogenic product according to claim 14,characterized in that the carrier protein molecule (ii) is selectedamongst gp160, p24, p17, Nef and Tat proteins of the HIV1 virus.
 17. Animmunogenic product according to claim 14, characterized in that thecarrier protein molecule (ii) is selected amongst CEA, p53, Di12, CaSm,OSA and ETS2 proteins.
 18. An immunogenic product according to claim 14,characterized in that the carrier protein molecule (ii) is selectedamongst allergenic proteins such Bet v 1, Der p 1 and Fel d
 1. 19. Animmunogenic product according to claim 18 characterized in that theallergenic proteins are selected amongst Bet v 1, Der p 1 and Fel d 1.20. An immunogenic product according claim 1, characterized in that itis selected amongst products comprising the following heterocomplexes,wherein the antigenic proteins (i), on the one hand, and the proteincarrier molecule (ii), on the other hand, are respectively: a) (i) IL-4and (ii) KLH; b) (i) alpha interferon and (ii) KLH; c) (i) VEGF and (ii)KLH; d) (i) IL-10 and (ii) KLH; e) (i) alpha interferon and (ii) gp 160of VIH1; f) (i) IL-4 and (ii) the Bet v 1 allergenic antigen; and g) (i)VEGF and (ii) the papillomavirus E7 protein; h) (i) the inactivated VIH1Tat protein and (ii) the VIH1 gp 120 protein; i) (i) an IgE isotypehuman antibody and (ii) the inactivated VIH1 Tat protein; j) (i) thericin β fragment and (ii) KLH.
 21. A composition comprising animmunogenic product according to claim
 1. 22. A pharmaceuticalcomposition comprising an immunogenic product according to claim 1 inassociation with one or more physiologically compatible excipients. 23.An immunogenic composition comprising an immunogenic product accordingto claim 1 in association with one or more physiologically compatibleexcipients.
 24. A vaccine composition comprising an immunogenic productaccording to claim 1 in association with one or more physiologicallycompatible excipients.
 25. An immunogenic composition or a vaccinecomposition according to claim 23, characterized in that it comprisesthe CpG immunity adjuvant.
 26. A method for preparing an immunogenicproduct according to claim 1, characterized in that it comprises thefollowing steps of: a) incubating the antigenic proteins (i) and thecarrier molecule (ii) in a molar ratio (i):(ii) ranging from 10:1 to50:1 in the presence of a chemical binding agent; b) collecting theimmunogenic product comprising immunogenic heterocomplexes beingprepared in step a).
 27. A method according to claim 26, characterizedin that the chemical binding agent is glutaraldehyde.
 28. A methodaccording to claim 26, characterized in that step a) is followed by astabilizing step of the immunogenic heterocomplexes by the formaldehyde,prior to the step b) of collecting the immunogenic product.